Rice promoters for regulation of plant expression

ABSTRACT

The present invention provides promoters from plants capable of driving gene expression in plant cells. The promoters vary in strength and in tissue specificity, and can be used to facilitate the development of transgenic plants in which tissue preferred expression, constitutive expression, and the strength of transgene expression is either more or less critical.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.12/395,171, filed on Feb. 27, 2009, which is a divisional of U.S. patentapplication Ser. No. 11/253,199, filed Oct. 18, 2005, which is acontinuation-in-part of U.S. patent application Ser. No. 10/260,238filed Sep. 26, 2002 (now abandoned), which itself claims the benefit ofU.S. Provisional Application No. 60/325,448, filed Sep. 26, 2001, U.S.Provisional Application No. 60/325,277 filed Sep. 26, 2001, and U.S.Provisional Application No. 60/370,620 filed Apr. 4, 2002. Thedisclosures of all of these applications are incorporated herein byreference in their entireties.

FIELD OF THE INVENTION

The present invention relates generally to the field of plant molecularbiology. More specifically, it relates to the regulation of geneexpression in plants such as monocots.

BACKGROUND OF THE INVENTION

Manipulation of crop plants to alter and/or improve phenotypiccharacteristics (such as productivity or quality) requires theexpression of heterologous genes in plant tissues. Such geneticmanipulation relies on the availability of a means to drive and tocontrol gene expression as required. For example, genetic manipulationrelies on the availability and use of suitable promoters which areeffective in plants and which regulate gene expression so as to give thedesired effect(s) in the transgenic plant. It is advantageous to havethe choice of a variety of different promoters so that the most suitablepromoter may be selected for a particular gene, construct, cell, tissue,plant or environment. Moreover, the increasing interest incotransforming plants with multiple plant transcription units (PTU) andthe potential problems associated with using common regulatory sequencesfor these purposes merit having a variety of promoter sequencesavailable.

There is, therefore, a great need in the art for the identification ofnovel sequences that can be used for expression of selected transgenesin economically important plants. More specifically, there is a need forthe systematic identification of genes that are expressed in aparticular manner, e.g., using microarray technology.

SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid molecule(polynucleotide) having a plant nucleotide sequence that directstissue-specific or tissue-preferential, or constitutive, transcriptionof a linked nucleic acid segment in a plant or plant cell, e.g., alinked plant DNA comprising an open reading frame for a structural orregulatory gene.

In one embodiment of the invention, the nucleotide sequence of theinvention directs tissue-specific (or tissue-preferential), orconstitutive, transcription of a linked nucleic acid segment in a plantor plant cell and is preferably obtained or obtainable from plantgenomic DNA having a gene comprising an open reading frame (ORF)encoding a polypeptide which is substantially similar, and preferablyhas at least 70% or more, e.g., between 71% and 89%, and even 90% ormore, e.g., between 91% and 99%, amino acid sequence identity, to apolypeptide encoded by an Oryza, e.g., Oryza sativa, gene, with eachindividual number within this range of between 71% and 89% and 91% and99% also being part of the invention, wherein said gene comprises anyone of:

-   -   (i) SEQ ID NOs:2275-2672, 5959, 5972, 5973, 5977-5990 and 6001        (e.g., including a promoter obtained or obtainable from any one        of SEQ ID NOs:2275-2672, 5959, 5972, 5973, 5977-5990 and 6001)        which directs seed-specific (or seed-preferential) transcription        of a linked nucleic acid segment;    -   (ii) SEQ ID NOs:2144-2274 (e.g., including a promoter obtained        or obtainable from any one of SEQ ID NOs:2144-2274) which        directs root-specific (or root-preferential) transcription of a        linked nucleic acid segment;    -   (iii) SEQ ID NOs:1886-1918 (e.g., including a promoter obtained        or obtainable from any one of SEQ ID NOs:1886-1918) which        directs green tissue (leaf and stem)-specific (or green        tissue-preferential) transcription of a linked nucleic acid        segment;    -   (iv) SEQ ID NOs:1919-2085 (e.g., including a promoter obtained        or obtainable from any one of SEQ ID NOs:1919-2085) which        directs panicle-specific (or panicle-preferential) transcription        of a linked nucleic acid segment;    -   (v) SEQ ID NOs:2086-2143 (e.g., including a promoter obtained or        obtainable from any one of SEQ ID NOs:2086-2143) which directs        pollen-specific (or pollen-preferential) transcription of a        linked nucleic acid segment;    -   (vi) SEQ ID NOs: 1598-1885 and 5960-5971 (e.g., including a        promoter obtained or obtainable from any one of SEQ ID NOs:        1598-1885 and 5960-5971, respectively) which directs        constitutive transcription of a linked nucleic acid segment;        or    -   (a) a fragment (portion) thereof which has substantially the        same promoter activity as the corresponding promoter listed in        SEQ ID NOs:2275-2672, 5959, 5972, 5973, 5977-5990 and 6001, SEQ        ID NOs:2144-2274, SEQ ID NOs:1886-1918, SEQ ID NOs:1919-2085, or        SEQ ID NOs: 1598-1885 and 5960-5971;    -   (b) a nucleotide sequence having substantial similarity to a        promoter sequence listed in SEQ ID NOs:2275-2672, 5959, 5972,        5973, 5977-5990 and 6001, SEQ ID NOs:2144-2274, SEQ ID        NOs:1886-1918, SEQ ID NOs:1919-2085, or SEQ ID NOs: 1598-1885        and 5960-5971;    -   (c) a nucleotide sequence capable of hybridizing to a promoter        sequence listed in SEQ ID NOs:2275-2672, 5959, 5972, 5973,        5977-5990 and 6001, SEQ ID NOs:2144-2274, SEQ ID NOs:1886-1918,        SEQ ID NOs:1919-2085, or SEQ ID NOs: 1598-1885 and 5960-5971;    -   (d) a nucleotide sequence capable of hybridizing to a nucleic        acid comprising 50 to 200 or more consecutive nucleotides of a        nucleotide sequence listed in SEQ ID NOs:2275-2672, 5959, 5972,        5973, 5977-5990 and 6001, SEQ ID NOs:2144-2274, SEQ ID        NOs:1886-1918, SEQ ID NOs:1919-2085, or SEQ ID NOs: 1598-1885        and 5960-5971 or the complement thereof;    -   (e) a nucleotide sequence which is the complement or reverse        complement of any of the previously mentioned nucleotide        sequences.

For example, in one embodiment, a plant nucleotide sequence is thepromoter sequence for a gene, and preferably is obtained or obtainablefrom a gene, comprising an ORF encoding a polypeptide which issubstantially similar, and preferably has at least 70% or more, e.g.,between 71% and 89%, and even 90% or more, e.g., between 91% and 99%,amino acid sequence identity, to a polypeptide encoded by an Oryza,e.g., Oryza sativa, gene, with each individual number within this rangeof between 71% and 89% and 91% and 99% also being part of the invention,wherein said gene comprises an ORF comprising a nucleic acid sequenceselected from the group consisting of SEQ ID NOs: 1-398 and 5928-5939(constitutively expressed ORFs), SEQ ID NOs:399-464 (green-specificORFs); SEQ ID NOs:465-720 (panicle-specific ORFs), SEQ ID NOs:721-800(pollen-specific ORFs), SEQ ID NOs:801-1019 (root-specific ORFs), SEQ IDNOs:1020-1597, 5927, 5940, 5941, 5945-5958 (seed-specific ORFs), and afragment (portion) thereof which encodes a polypeptide which hassubstantially the same activity as the corresponding polypeptide encodedby an ORF listed in SEQ ID NOs: 1-398 and 5928-5939; SEQ ID NOs:399-464, SEQ ID NOs:465-720, SEQ ID NOs:721-800, SEQ ID NOs:801-1019,and SEQ ID NOs:1020-1597, 5927, 5940, 5941, 5945-5958.

In another embodiment, a plant nucleotide sequence is the promotersequence for a gene, and preferably is obtained or obtainable from agene, which is substantially similar, and preferably has at least 70%,or more, e.g., between 71% and 89%, and even 90% or more, e.g., between91% and 99%, nucleic acid sequence identity to an Oryza gene, with eachindividual number within this range of between 71% and 89% and 91% and99% also being part of the invention, wherein said gene comprises anucleic acid sequence selected from the group consisting of SEQ IDNOs:2275-2672, 5959, 5972, 5973, 5977-5990 and 6001, SEQ IDNOs:2144-2274, SEQ ID NOs:1886-1918, SEQ ID NOs:1919-2085, SEQ IDNOs:2086-2143, SEQ ID NOs: 1598-1885 and 5960-5971, and a fragment(portion) thereof which has substantially the same promoter activity asthe corresponding promoter listed in SEQ ID NOs:2275-2672, 5959, 5972,5973, 5977-5990 and 6001, SEQ ID NOs:2144-2274, SEQ ID NOs:1886-1918,SEQ ID NOs:1919-2085, SEQ ID NOs:2086-2143, and SEQ ID NOs: 1598-1885and 5960-5971.

In another embodiment the invention relates to a nucleotide sequence fora promoter, which is preferably obtained or obtainable from plantgenomic DNA, from a gene comprising an ORF which is substantiallysimilar, and preferably has at least 70% or more, e.g., between 71% and89%, and even 90% or more, e.g., between 91% and 99%, nucleic acidsequence identity, to an Oryza gene, with each individual number withinthis range of between 71% and 89% and 91% and 99% also being part of theinvention, wherein said gene comprises an ORF comprising one of thesequences selected from the group consisting of SEQ ID NOs: 1-398 and5928-5939; SEQ ID NOs: 399-464, SEQ ID NOs:465-720, SEQ ID NOs:721-800,SEQ ID NOs:801-1019, SEQ ID NOs:1020-1597, 5927, 5940, 5941, 5945-5958,and a fragment (portion) thereof which encodes a polypeptide which hassubstantially the same activity as the corresponding polypeptide encodedby an ORF listed in SEQ ID NOs: 1-398; SEQ ID NOs: 399-464, SEQ IDNOs:465-720, SEQ ID NOs:721-800, SEQ ID NOs:801-1019, and SEQ IDNOs:1020-1597, 5927, 5940, 5941, 5945-5958.

Hence, the isolated nucleic acid molecules of the invention include theorthologs of the Oryza sequences disclosed herein, i.e., thecorresponding nucleotide sequences in organisms other than Oryza,including, but not limited to, plants other than Oryza, preferablycereal plants, e.g., corn, wheat, rye, turfgrass, sorghum, millet,sugarcane, barley and banana, but also non-cereal plants, e.g., alfalfa,sunflower, canola, soybean, cotton, peanut, tobacco or sugarbeet. Anorthologous gene is a gene from a different species that encodes aproduct having the same or similar function, e.g., catalyzing the samereaction as a product encoded by a gene from a reference organism. Thus,an ortholog includes polypeptides having less than, e.g., 65% amino acidsequence identity, but which ortholog encodes a polypeptide having thesame or similar function. Databases such GenBank may be employed toidentify sequences related to the Oryza sequences, e.g., orthologs incereal crops such as wheat and other cereals. Alternatively, recombinantDNA techniques such as hybridization or PCR may be employed to identifysequences related to the Oryza sequences or to clone the equivalentsequences from different Oryza DNAs. For example, SEQ ID NOs:2673-4708,SEQ ID NOs: 4768-5229, and SEQ ID NOs:5230-5926, which represent wheat,banana and maize orthologs of some of the rice sequences disclosedherein. The encoded ortholog products likely have at least 70% sequenceidentity to each other. Hence, the invention includes an isolatednucleic acid molecule comprising a nucleotide sequence from a gene thatencodes a polypeptide having at least 70% identity to a polypeptideencoded by a gene having one or more of the Oryza sequences disclosedherein. For example, promoter sequences within the scope of theinvention are those which direct expression of an open reading framewhich encodes a polypeptide that is substantially similar to an Oryzapolypeptide encoded by a gene having a promoter selected from the groupconsisting of SEQ ID NOs:1598-2672, 5959, 5972, 5973, 5977-5990 and6001.

Preferably, the promoters of the invention include a consecutive stretchof about 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000or 1500, contiguous nucleotides, e.g., 40 to about 743, 60 to about 743,125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743,of any one of SEQ ID NOs:1598-2672, 5959, 5972, 5973, 5977-5990 and6001, or the promoter orthologs thereof, which include the minimalpromoter region.

In a particular embodiment of the invention said consecutive stretch ofabout 25 to 2000, including 50 to 500 or 100 to 250, and up to 1000 or1500, contiguous nucleotides, e.g., 40 to about 743, 60 to about 743,125 to about 743, 250 to about 743, 400 to about 743, 600 to about 743,has at least 75%, preferably 80%, more preferably 90% and mostpreferably 95%, nucleic acid sequence identity with a correspondingconsecutive stretch of about 25 to 2000, including 50 to 500 or 100 to250, and up to 1000 or 1500, contiguous nucleotides, e.g., 40 to about743, 60 to about 743, 125 to about 743, 250 to about 743, 400 to about743, 600 to about 743, of any one of SEQ ID NOs:1598-2672, 5959, 5972,5973, 5977-5990 and 6001, or the promoter orthologs thereof, whichinclude the minimal promoter region. The above defined stretch ofcontiguous nucleotides preferably comprises one or more promoter motifsselected from the group consisting of TATA box, GC-box, CAAT-box and atranscription start site.

In case of promoters directing tissue-specific transcription of a linkednucleic acid segment in a plant or plant cell such as, for example, apromoter directing root-specific, green tissue (leaf and stem)-specific,seed-specific, panicle-specific, pollen-specific, etc., transcription,it is further preferred that previously defined stretch of contiguousnucleotides comprises further motifs that participate in the tissuespecificity of said stretch(es) of nucleotides, e.g., for seed-specificpromoters, motifs selected from the group consisting of the P box andGCNA elements, including but not limited to TGTAAAG and TGA(G/C)TCA.

The invention also provides an isolated nucleic acid molecule, e.g., DNAor RNA, comprising a plant nucleotide sequence comprising an openreading frame that is preferentially expressed in a specific planttissue, i.e., in seeds, roots, green tissue (leaf and stem), panicle orpollen, or is expressed constitutively.

One embodiment the invention provides

-   -   (a) an isolated nucleic acid molecule, e.g., DNA or RNA,        comprising a plant nucleotide sequence comprising an ORF that is        constitutively expressed or preferentially expressed in a        specific plant tissue, i.e., in seeds, roots, green tissue (leaf        and stem), panicle or pollen and which is capable of hybridizing        and thus substantially similar, and preferably has at least 70%        or more, e.g., between 71% and 89%, and even 90% or more, e.g.,        between 91% and 99%, nucleic acid sequence identity, to an ORF        expressed in a constitutive (e.g., an ORF comprising one of SEQ        ID NOs:1-398 and 5928-5939) or in a tissue-specific or        tissue-preferential manner, for example, in    -   a seed-specific (or seed-preferential) manner, e.g., an ORF        comprising one of SEQ ID NOs:1020-1597; 5927, 5940, 5941,        5945-5958        -   (i) a root-specific (or root-preferential) manner, e.g., an            ORF comprising one of SEQ ID NOs:801-1019;        -   (ii) a green tissue (leaf and stem)-specific (or green            tissue (leaf and stem)-preferential) manner, e.g., an ORF            comprising one of SEQ ID NOs:399-464;        -   (iii) a panicle-specific (or panicle-preferential) manner,            e.g., an ORF comprising one of SEQ ID NOs:465-720; or        -   (iv) a pollen-specific (or pollen-preferential) manner,            e.g., an ORF comprising one of SEQ ID NOs:721-800; or    -   (b) a part thereof still encoding a partial-length polypeptide        having substantially the same activity as the full-length        polypeptide encoded by an ORF listed in SEQ ID NOs.1-398, and        5928-5939 and 399-1597, 5927, 5940, 5941, 5945-5958., e.g., at        least 50%, more preferably at least 80%, even more preferably at        least 90% to 95% the activity of the full-length polypeptide;    -   (c) the complement or reverse complement thereof

The invention also provides

-   -   (a) an isolated nucleic acid molecule, e.g., DNA or RNA,        comprising a plant nucleotide sequence comprising an ORF that is        constitutively expressed or preferentially expressed in a        specific plant tissue, i.e., in seeds, roots, green tissue (leaf        and stem), panicles or pollen and which encodes a polypeptide        that is capable of hybridising and thus substantially similar,        and preferably has at least 70% or more, e.g., between 71% and        89%, and even 90% or more, e.g., between 91% and 99%, amino acid        sequence identity, to a polypeptide encoded by an Oryza gene        with each individual number within this range of between 71% and        89% and 91% and 99% also being part of the invention, wherein        said gene comprises an ORF comprising any one of the sequences        selected from the group consisting of SEQ ID NOs: 1-398, and        5928-5939 (constitutiv); SEQ ID NOs: 399-464 (green-tissue), SEQ        ID NOs:465-720 (specific); SEQ ID NOs:721-800 (pollen); SEQ ID        NOs:801-1019 (root); and SEQ ID NOs:1026-1597, 5927, 5940, 5941,        5945-5958 (seed),    -   (b) the complement or reverse complement thereof, and    -   (c) a fragment thereof still encoding a partial-length        polypeptide having substantially the same activity as the        full-length polypeptide encoded by an ORF listed in SEQ ID        NOs.1-398 and 5928-5939 and 399-1597, 5927, 5940, 5941,        5945-5958, e.g., at least 50%, more preferably at least 80%,        even more preferably at least 90% to 95% the activity of the        full-length polypeptide

The invention also provides

-   -   (a) an isolated nucleic acid molecule, e.g., DNA or RNA,        comprising a plant nucleotide sequence comprising an ORF that is        constitutively expressed or preferentially expressed in a        specific plant tissue, i.e., in seeds, roots, green tissue (leaf        and stem), panicles or pollen and which encodes a polypeptide        that is capable of hybridizing and thus substantially similar,        and preferably has at least 70% or more, e.g., between 71% and        89%, and even 90% or more, e.g., between 91% and 99%, amino acid        sequence identity, to a polypeptide encoded by an Oryza gene        with each individual number within this range of between 71% and        89% and 91% and 99% also being part of the invention, wherein        said gene comprises a promoter sequence as given in any one of        the sequences selected from the group consisting of SEQ ID NOs:        1598-1885 and 5960-5971, SEQ ID NOs: 1886-1918, SEQ ID        NOs:1919-2085, SEQ ID NOs:2086-2143, SEQ ID NOs:2144-2274, and        SEQ ID NOs:2275-2672, 5959, 5972, 5973, 5977-5990 and 6001    -   (b) the complement or reverse complement thereof, and    -   (c) a fragment thereof having substantially the same activity as        the corresponding promoter listed in SEQ ID NOs: SEQ ID NOs:        1598-1885, 5960-5971 and 1886-2672, 5959, 5972, 5973, 5977-5990        and 6001 respectively, e.g., at least 50%, more preferably at        least 80%, even more preferably at least 90% to 95% of the        activity.

The invention also provides

-   -   (a) an isolated nucleic acid molecule, e.g., DNA or RNA,        comprising a plant nucleotide sequence comprising an ORF that is        constitutively or preferentially expressed in a specific plant        tissue, i.e., in seeds, roots, green tissue (leaf and stem),        panicles or pollen and which is capable of hybridizing and thus        substantially similar, and preferably has at least 70% or more,        e.g., between 71% and 89%, and even 90% or more, e.g., between        91% and 99%, nucleic acid sequence identity, to an Oryza gene        with each individual number within this range of between 71% and        89% and 91% and 99% also being part of the invention, wherein        said gene is an ORF expressed in a constitutive or a        tissue-specific or tissue-preferential manner and comprises a        promoter as given in any one of the sequences selected from the        group consisting of SEQ ID NOs: 1598-1885 and 5960-5971; SEQ ID        NOs: 1886-1918, SEQ ID NOs:1919-2085; SEQ ID NOs:2086-2143; SEQ        ID NOs:2144-2274; and SEQ ID NOs:2275-2672, 5959, 5972, 5973,        5977-5990 and 6001    -   (b) the complement or reverse complement thereof, and    -   (c) and a fragment thereof having substantially the same        activity as the corresponding promoter listed in SEQ ID NOs:        1598-1885, 5960-5971 and SEQ ID NOs:1886-2672, 5959, 5972, 5973,        5977-5990 and 6001 respectively, e.g., at least 50%, more        preferably at least 80%, even more preferably at least 90% to        95% of the activity.

ORFs which are expressed in a constitutive or in tissue-specific or-preferential manner, may be useful to prepare plants that over- orunder-express the encoded polypeptide product or to prepare knockoutplants.

The promoters and open reading frames of the invention can be identifiedby employing an array of nucleic acid samples, e.g., each sample havinga plurality of oligonucleotides, and each plurality corresponding to adifferent plant gene, on a solid substrate, e.g., a DNA chip, and probescorresponding to nucleic acid expressed in, for example, one or moreplant tissues and/or at one or more developmental stages, e.g., probescorresponding to nucleic acid expressed in seed of a plant relative tocontrol nucleic acid from sources other than seed. Thus, genes that areupregulated or downregulated in the majority of tissues at a majority ofdevelopmental stages, or upregulated or downregulated in one tissue suchas in seed, can be systematically identified.

As described herein, GENECHIP® technology was utilized to discover ricegenes that are preferentially (or exclusively) expressed in seed,pollen, specific, root or green tissue, as well as those that areconstitutively expressed. Specifically, labeled rice cRNA probes werehybridized to the rice DNA array, expression levels were determined bylaser scanning and then rice genes were identified that had a particularexpression pattern. The rice oligonucleotide probe array consists ofprobes from over 18,000 unique rice genes, which covers approximately40-50% of the genome. This genome array permits a broader, more completeand less biased analysis of gene expression. Using this approach, 812genes were identified, the expression of which was altered, e.g.,specifically elevated, in seed tissues and 367 genes were identifiedthat were preferentially expressed in endosperm, 91 genes wereidentified that were preferentially expressed in embryo, and 137 geneswere identified that were preferentially expressed in aleurone; 618genes were identified that were constitutively expressed; 335 genes wereidentified that were specifically or preferentially expressed inpanicle; 265 genes were identified that were specifically orpreferentially expressed in root tissue, 80 genes were identified thatwere specifically or preferentially expressed in pollen; and 90 geneswere identified that were specifically or preferentially expressed inleaf and/or stem tissue.

Generally, the promoters of the invention may be employed to express anucleic acid segment that is operably linked to said promoter such as,for example, an open reading frame, or a portion thereof, an anti-sensesequence, or a transgene in plants. The open reading frame may beobtained from an insect resistance gene, a disease resistance gene suchas, for example, a bacterial disease resistance gene, a fungal diseaseresistance gene, a viral disease resistance gene, a nematode diseaseresistance gene, a herbicide resistance gene, a gene affecting graincomposition or quality, a nutrient utilization gene, a mycotoxinreduction gene, a male sterility gene, a selectable marker gene, ascreenable marker gene, a negative selectable marker, a positiveselectable marker, a gene affecting plant agronomic characteristics,i.e., yield, standability, and the like, or an environment or stressresistance gene, i.e., one or more genes that confer herbicideresistance or tolerance, insect resistance or tolerance, diseaseresistance or tolerance (viral, bacterial, fungal, oomycete, ornematode), stress tolerance or resistance (as exemplified by resistanceor tolerance to drought, heat, chilling, freezing, excessive moisture,salt stress, or oxidative stress), increased yields, food content andmakeup, physical appearance, male sterility, drydown, standability,prolificacy, starch properties or quantity, oil quantity and quality,amino acid or protein composition, and the like. By “resistant” is meanta plant which exhibits substantially no phenotypic changes as aconsequence of agent administration, infection with a pathogen, orexposure to stress. By “tolerant” is meant a plant which, although itmay exhibit some phenotypic changes as a consequence of infection, doesnot have a substantially decreased reproductive capacity orsubstantially altered metabolism.

In particular, seed-specific promoters may be useful for expressinggenes as well as for producing large quantities of protein, forexpressing oils or proteins of interest, e.g., antibodies, genes forincreasing the nutritional value of the seed and the like.Panicle-specific, root-specific, and pollen-specific promoters may beuseful for expressing genes that confer pathogen-resistance, e.g.,insect resistance, to those tissues, or to silence other genes that areexpressed in those tissues.

For instance, pollen-specific promoters may be employed to introducegenes into pollen for the purpose of arresting pollen developmentthereby rendering a plant male sterile. Such genes may include thosecoding for proteins toxic to pollen. It is also contemplated thatchimeric plasmids may be constructed which allow the expression ofantisense mRNAs which are capable of inhibiting expression of geneswhich play a role in pollen development. It is also contemplated thatexpression cassettes or vectors of the present invention which comprisea pollen-specific promoter may be useful for the introduction of one ormore useful phenotypic characteristics into pollen including but notlimited to pesticide resistance, resistance to insect pests or toxicityto insect pests, or which optimize other pollen functions. Oneembodiment the invention comprises genetic manipulation of plants topotentiate the effects of gibberellin or other hormones involved ininitiation of fruit set. The invention comprises the temporal expressionof a structural gene which encodes a plant hormone such as a gibberellinor cytokine, or proteins associated with the production of such hormones(i.e., enzymes, biosynthetic intermediates and the like.) which areassociated with initiation of fruit set. The structural gene is placedunder the control of a pollen microspore- or megaspore-specific promotersuch that the expression of the hormone is timed to occur just prior topollination so that fruit development and maturation is induced withoutthe need for fertilization.

Root-specific promoters may be useful for expressing genes including butnot limited to defense-related genes, including genes conferringinsecticidal resistance and stress tolerance, e.g., salt, cold ordrought tolerance, genes for altering nutrient uptake and genes that areinvolved with specific morphological traits that allow for increasedwater absorption, uptake or extraction from soil, e.g., soil of lowmoisture content. For example, introduction and expression of genes thatalter root characteristics may enhance water uptake. Additionally, theuse of root-specific promoters in transgenic plants can providebeneficial traits that are localized in the consumable (by animals andhumans) roots of plants such as carrots, parsnips, and beets. However,other parts of the plants, including stalks, husks, vegetative parts,and the like, may also be desirable, including use as part of animalsilage or for ornamental purposes. Often chemical constituents (e.g.,oils or starches) of maize and other crops are extracted for foods orindustrial use and transgenic plants may be created which have enhancedor modified levels of such components.

Green tissue-specific promoters may be useful for expressing genesincluding but not limited to genes involved in photosynthetic pathways,and for those which are leaf-specific, for producing large quantities ofprotein, and for expressing oils or proteins of interest, genes forincreasing the nutritional value of a plant, and defense-related genes(e.g., against pathogens such as a virus or fungus), including genesencoding insecticidal polypeptides.

Panicle-specific promoters may be useful for expressing genes includingbut not limited to genes involved in flower development and floweringsuch as MADS-box genes that, when expressed in transgenic plants, resultin such phenotypes as, for example, reduced apical dominance or dwarfismand early flowering.

Constitutive promoters are useful for expressing a wide variety of genesincluding those which alter metabolic pathways, confer diseaseresistance, for protein production, e.g., antibody production, or toimprove nutrient uptake and the like. Constitutive promoters may bemodified so as to be regulatable, e.g., inducible. The genes andpromoters described hereinabove can be used to identify orthologousgenes and their promoters which are also likely expressed in aparticular tissue and/or development manner. Moreover, the orthologouspromoters are useful to express linked open reading frames. In addition,by aligning the promoters of these orthologs, novel cis elements can beidentified that are useful to generate synthetic promoters.

The present invention further provides a composition, an expressioncassette or a recombinant vector containing the nucleic acid molecule ofthe invention, and host cells comprising the expression cassette orvector, e.g., comprising a plasmid. In particular, the present inventionprovides an expression cassette or a recombinant vector comprising apromoter of the invention linked to a nucleic acid segment which, whenpresent in a plant, plant cell or plant tissue, results in transcriptionof the linked nucleic acid segment. The invention also provides anexpression cassette or a recombinant vector comprising a plantnucleotide sequence comprising an open reading frame of the inventionwhich, when present in a plant, plant cell or plant tissue, results inexpression of the product encoded by the open reading frame. Further,the invention provides isolated polypeptides encoded by any one of theopen reading frames comprising SEQ ID NOs:1-1597, 5927, 5940, 5941,5945-5958, a fragment thereof which encodes a polypeptide which hassubstantially the same activity as the corresponding polypeptide encodedby an ORF listed in SEQ ID NOs:1-1597, 5927, 5940, 5941, 5945-5958, orthe orthologs thereof.

The invention also provides sense and anti-sense nucleic acid moleculescorresponding to the open reading frames identified in SEQ IDNOs:1-1597, 5927, 5940, 5941, 5945-5958 as well as their orthologs. Alsoprovided are compositions, expression cassettes, e.g., recombinantvectors, and host cells, comprising a nucleic acid molecule whichcomprises a nucleic acid segment which is preferentially expressed inseeds (e.g., SEQ ID NOs:1020-1597, 5927, 5940, 5941, 5945-5958), root(SEQ ID NOs:801-1019), pollen (SEQ ID NOs:721-800), specific (SEQ IDNOs:465-720), or green tissue (SEQ ID NOs:399-464), or constitutivelyexpressed (SEQ ID NOs:1-398 and 5928-5939), in either sense or antisenseorientation.

In one embodiment, the invention provides an expression cassette orvector containing an isolated nucleic acid molecule having a nucleotidesequence that directs tissue-specific, tissue-preferential orconstitutive transcription of a linked nucleic acid segment in a cell,which nucleotide sequence is from a gene which encodes a polypeptidehaving at least 70% identity to an Oryza polypeptide encoded by a genehaving one of the promoters listed in SEQ ID NOs:1598-2672, 5959, 5972,5973, 5977-5990 and 6001, and which nucleotide sequence is optionallyoperably linked to other suitable regulatory sequences, e.g., atranscription terminator sequence, operator, repressor binding site,transcription factor binding site and/or an enhancer. This expressioncassette or vector may be contained in a host cell. The expressioncassette or vector may augment the genome of a transformed plant or maybe maintained extrachromosomally. The expression cassette may beoperatively linked to a structural gene, the open reading frame thereof,or a portion thereof. The expression cassette may further comprise a Tiplasmid and be contained in an Agrobacterium tumefaciens cell; it may becarried on a microparticle, wherein the microparticle is suitable forballistic transformation of a plant cell; or it may be contained in aplant cell or protoplast. Further, the expression cassette or vector canbe contained in a transformed plant or cells thereof, and the plant maybe a dicot or a monocot. In particular, the plant may be a cereal plant.

The present invention further provides a method of augmenting a plantgenome by contacting plant cells with a nucleic acid molecule of theinvention, e.g., one having a nucleotide sequence that directstissue-specific, tissue-preferential or constitutive transcription of alinked nucleic acid segment isolatable or obtained from a plant geneencoding a polypeptide that is substantially similar to a polypeptideencoded by the an Oryza gene having a sequence according to any one ofSEQ ID NOs:1-2672, 5959, 5972, 5973, 5977-5990 and 6001 so as to yieldtransformed plant cells; and regenerating the transformed plant cells toprovide a differentiated transformed plant, wherein the differentiatedtransformed plant expresses the nucleic acid molecule in the cells ofthe plant. The nucleic acid molecule may be present in the nucleus,chloroplast, mitochondria and/or plastid of the cells of the plant. Thepresent invention also provides a transgenic plant prepared by thismethod, a seed from such a plant and progeny plants from such a plantincluding hybrids and inbreds. Preferred transgenic plants aretransgenic maize, soybean, barley, alfalfa, sunflower, canola, soybean,cotton, peanut, sorghum, tobacco, sugarbeet, rice, wheat, rye,turfgrass, millet, sugarcane, tomato, or potato.

A transformed (transgenic) plant of the invention includes plants, thegenome of which is augmented by a nucleic acid molecule of theinvention, or in which the corresponding gene has been disrupted, e.g.,to result in a loss, a decrease or an alteration, in the function of theproduct encoded by the gene, which plant may also have increased yieldsand/or produce a better-quality product than the corresponding wild-typeplant. The nucleic acid molecules of the invention are thus useful fortargeted gene disruption, as well as markers and probes.

The invention also provides a method of plant breeding, e.g., to preparea crossed fertile transgenic plant. The method comprises crossing afertile transgenic plant comprising a particular nucleic acid moleculeof the invention with itself or with a second plant, e.g., one lackingthe particular nucleic acid molecule, to prepare the seed of a crossedfertile transgenic plant comprising the particular nucleic acidmolecule. The seed is then planted to obtain a crossed fertiletransgenic plant. The plant may be a monocot or a dicot. In a particularembodiment, the plant is a cereal plant.

The crossed fertile transgenic plant may have the particular nucleicacid molecule inherited through a female parent or through a maleparent. The second plant may be an inbred plant. The crossed fertiletransgenic may be a hybrid. Also included within the present inventionare seeds of any of these crossed fertile transgenic plants.

The present invention also provides a method to identify a nucleotidesequence that directs tissue-specific or tissue-preferentialtranscription of linked nucleic acid in the genome of a plant cell bycontacting a probe of plant nucleic acid, e.g., cRNA from rice, isolatedfrom various tissues of a plant, with a plurality of isolated nucleicacid samples on one or more, i.e., a plurality of, solid substrates soas to form a complex between at least a portion of the probe and anucleic acid sample(s) having sequences that are structurally related tothe sequences in the probe. Each sample comprises one or a plurality ofoligonucleotides corresponding to at least a portion of a plant gene.Then complex formation is compared between samples contacted with aparticular tissue, e.g., a seed-specific, probe and samples contactedwith a different tissue, e.g., a non-seed specific probe, so as todetermine which RNAs are expressed in the particular tissue of theplant. The probe and/or samples may be nucleic acid from a dicot or froma monocot.

The present invention also provides a method to identify a nucleotidesequence that directs constitutive transcription of nucleic acid in thegenome of a plant cell by contacting a probe of plant nucleic acid,e.g., cRNA from rice, isolated from various tissues of a plant and atvarious developmental stages with a plurality of isolated nucleic acidsamples on one or more, i.e., a plurality of, solid substrates so as toform a complex between at least a portion of the probe and a nucleicacid sample(s) having sequences that are structurally related to thesequences in the probe. Each sample comprises one or a plurality ofoligonucleotides corresponding to at least a portion of a plant gene.Complex formation is then compared to determine which RNAs are presentin a majority of, preferably in substantially all, tissues, in amajority of, preferably at substantially all, developmental stages ofthe plant. The probe and/or samples may be nucleic acid from a dicot orfrom a monocot.

The compositions of the invention include plant nucleic acid molecules,and the amino acid sequences for the polypeptides or partial-lengthpolypeptides encoded by the nucleic acid molecule which comprises anopen reading frame. These sequences can be employed to alter expressionof a particular gene corresponding to the open reading frame bydecreasing or eliminating expression of that plant gene or byoverexpressing a particular gene product. Methods of this embodiment ofthe invention include stably transforming a plant with the nucleic acidmolecule which includes an open reading frame operably linked to apromoter capable of driving expression of that open reading frame (senseor antisense) in a plant cell. By “portion” or “fragment”, as it relatesto a nucleic acid molecule which comprises an open reading frame or afragment thereof encoding a partial-length polypeptide having theactivity of the full length polypeptide, is meant a sequence having atleast 80 nucleotides, more preferably at least 150 nucleotides, andstill more preferably at least 400 nucleotides. If not employed forexpressing, a “portion” or “fragment” means at least 9, preferably 12,more preferably 15, even more preferably at least 20, consecutivenucleotides, e.g., probes and primers (oligonucleotides), correspondingto the nucleotide sequence of the nucleic acid molecules of theinvention. Thus, to express a particular gene product, the methodcomprises introducing to a plant, plant cell, or plant tissue anexpression cassette comprising a promoter linked to an open readingframe so as to yield a transformed differentiated plant, transformedcell or transformed tissue. Transformed cells or tissue can beregenerated to provide a transformed differentiated plant. Thetransformed differentiated plant or cells thereof preferably expressesthe open reading frame in an amount that alters the amount of the geneproduct in the plant or cells thereof, which product is encoded by theopen reading frame. The present invention also provides a transformedplant prepared by the method, progeny and seed thereof.

The invention further includes a nucleotide sequence which iscomplementary to one (hereinafter “test” sequence) which hybridizesunder stringent conditions with a nucleic acid molecule of the inventionas well as RNA which is transcribed from the nucleic acid molecule. Whenthe hybridization is performed under stringent conditions, either thetest or nucleic acid molecule of invention is preferably supported,e.g., on a membrane or DNA chip. Thus, either a denatured test ornucleic acid molecule of the invention is preferably first bound to asupport and hybridization is effected for a specified period of time ata temperature of, e.g., between 55 and 70° C., in double strengthcitrate buffered saline (SC) containing 0.1% SDS followed by rinsing ofthe support at the same temperature but with a buffer having a reducedSC concentration. Depending upon the degree of stringency required suchreduced concentration buffers are typically single strength SCcontaining 0.1% SDS, half strength SC containing 0.1% SDS and one-tenthstrength SC containing 0.1% SDS.

A computer readable medium containing one or more of the nucleotidesequences of the invention as well as methods of use for the computerreadable medium are provided. This medium allows a nucleotide sequencecorresponding to at least one of SEQ ID NOs:1598-2672, 5959, 5972, 5973,5977-5990 and 6001 (promoters), SEQ ID NOs:1-1597, 5927, 5940, 5941,5945-5958 and 2673-5926 (orthologous open reading frames of wheat,banana and maizeor fragments thereof), to be used as a referencesequence to search against a database. This medium also allows forcomputer-based manipulation of a nucleotide sequence corresponding to atleast one of SEQ ID NOs:1-60001.

BRIEF DESCRIPTION OF THE TABLES

Table 1 provides the SEQ ID NOs and corresponding description for Oryzagenes which are expressed in a constitutive manner and further the SEQID NOs for the corresponding homologous sequences found in wheat, bananaand maize.

-   -   identifies a first subset of genes.    -   identifies a 2^(nd) subset of genes.

Three subgroups of constitutively expressed genes can be distinguishedbased on the expression level of those genes. The levels are ranked fromhighest (1) to lowest (3). For example, promoters with the highest levelof constitutive expression include those having an open reading framecorresponding to SEQ ID NOs:1-24, the next highest include those havingan open reading frame corresponding to SEQ ID NOs:25-142, the nexthighest include those having an open reading frame corresponding to SEQID NOs:143-293, and the lowest include those having an open readingframe corresponding to SEQ ID NOs:294-398 and 5928-5939.

Table 2 provides the SEQ ID NOs: and corresponding description for Oryzagenes which are expressed in a seed-specific manner and further the SEQID NOs for the corresponding homologous sequences found in wheat, bananaand maize.

Six subgroups of seed-specific genes can be distinguished based on theexpression level of those genes. The levels are ranked from highest (1)to lowest (6). For example, promoters with the highest level ofseed-specific expression include those from a gene having an openreading frame corresponding to SEQ ID NOs:1020-1021, the next highestinclude those from a gene having an open reading frame corresponding toSEQ ID NOs:1022-1025, the next highest include those from a gene havingan open reading frame corresponding to SEQ ID NOs:1026-1030, the nexthighest include those from a gene having an open reading framecorresponding to SEQ ID NOs:1031-1048, the next highest include thosefrom a gene having an open reading frame corresponding to SEQ IDNOs:1049-1165 and the lowest include those from a gene having an openreading frame corresponding to SEQ ID NOs:1166-1597, 5927, 5940, 5941,5945-5958.

Table 3 provides the SEQ ID NOs: and corresponding description for Oryzagenes which are expressed in an aleurone-specific manner and further theSEQ ID NOs for the corresponding homologous sequences found in wheat,banana and maize.

Table 4 provides the SEQ ID NOs: and corresponding description for Oryzagenes which are expressed in an endosperm-specific manner and furtherthe SEQ ID NOs for the corresponding homologous sequences found inwheat, banana and maize.

Table 5 provides the SEQ ID NOs: and corresponding description for Oryzagenes which are expressed in an embryo-specific manner and further theSEQ ID NOs for the corresponding homologous sequences found in wheat,banana and maize.

Table 6 provides the SEQ ID NOs: and corresponding description for Oryzagenes which are expressed in a leaf- and stem-specific manner andfurther the SEQ ID NOs for the corresponding homologous sequences foundin wheat, banana and maize.

Four subgroups of leaf- and stem-specific genes can be distinguishedbased on the expression level of those genes. The levels are ranked fromhighest (1) to lowest (4). For example, promoters with the highest levelof leaf and stem-specific expression include those from a gene having anopen reading frame corresponding to SEQ ID NOs:399-404, the next highestinclude those from a gene having an open reading frame corresponding toSEQ ID NOs:405-416, the next highest include those from a gene having anopen reading frame corresponding to SEQ ID NOs:417-456, and the lowestinclude those from a gene having an open reading frame corresponding toSEQ ID NOs:457-464.

Table 7 provides the SEQ ID NOs: and corresponding description for Oryzagenes which are expressed in a panicle-specific manner and further theSEQ ID NOs for the corresponding homologous sequences found in wheat,banana and maize.

Three subgroups of panicle-specific genes can be distinguished based onthe expression level of those genes. The levels are ranked from highest(1) to lowest (3). For example, promoters with the highest level ofpanicle-specific expression include those from a gene having an openreading frame corresponding to SEQ ID NOs:465-469, the next highestinclude those from a gene having an open reading frame corresponding toSEQ ID NOs:470-535, and the lowest include those from a gene having anopen reading frame corresponding to SEQ ID NOs:536-720.

Table 8 provides the SEQ ID NOs: and corresponding description for Oryzagenes which are expressed in a root-specific manner and further the SEQID NOs for the corresponding homologous sequences found in wheat, bananaand maize.

Four subgroups of root-specific genes can be distinguished based on theexpression level of those genes. The levels are ranked from highest (1)to lowest (4). For example, promoters with the highest level ofroot-specific expression include those from a gene having an openreading frame corresponding to SEQ ID NOs:801-809, the next highestinclude those from a gene having an open reading frame corresponding toSEQ ID NOs:810-846, the next highest include those from a gene having anopen reading frame corresponding to SEQ ID NOs:847-885, and the lowestinclude those from a gene having an open reading frame corresponding toSEQ ID NOs:886-1019.

Table 9 provides the SEQ ID NOs: and corresponding description for Oryzagenes which are express in a pollen-specific manner and further the SEQID NOs for the corresponding homologous sequences found in wheat, bananaand maize.

Three subgroups of pollen-specific genes can be distinguished based onthe expression level of those genes. The levels are ranked from highest(1) to lowest (3). For example, promoters with the highest level ofpollen-specific expression include those from a gene having an openreading frame corresponding to SEQ ID NOs:721-728, the next highestinclude those from a gene having an open reading frame corresponding toSEQ ID NOs:729-743, and the lowest include those from a gene having anopen reading frame corresponding to SEQ ID NOs:744-800.

Table 10 identifies the start and end point and the nucleotide sequencesof tri-nucleotide repeat units in the coding sequence of selected ORFs.

Table 11 provides Swiss Prot information.

Table 12 illustrates the promoter designation, probe set or gene, genedescription, PCR product size for a promoter containing PCR product andprimers employed to amplify promoter sequences, for exemplaryconstitutively expressed promoters.

Table 13 provides a listing of of Rice promoter sequences tested inExample 14

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the present invention, nucleic acid constructs areprovided that allow initiation of transcription in a “tissue-specific”,i.e., seed-, root-, green tissue (leaf and stem)-, panicle-, orpollen-specific, or in a constitutive manner. Constructs of theinvention comprise regulated transcription initiation regions associatedwith protein translation elongation, and the compositions of the presentinvention are drawn to novel nucleotide sequences for tissue-specific aswell as constitutive expression. The present invention thus provides forisolated nucleic acid molecules comprising a plant nucleotide sequencethat directs tissue-specific, i.e., seed-, root-, green tissue (leaf andstem)-, panicle-, or pollen-specific, transcription of a linked nucleicacid segment in a plant cell. Preferably, nucleotide sequence isobtained or obtainable from plant genomic DNA from a gene encoding apolypeptide which is substantially similar and preferably has at least70% amino acid sequence identity to a polypeptide encoded by an Oryzagene comprising any one of SEQ ID NOs:2275-2672, 5959, 5972, 5973,5977-5990 and 6001 (seed-specific promoters) and SEQ ID NOs:1020-1597,5927, 5940, 5941, 5945-5958 (seed-specific ORFs); SEQ ID NOs:2144-2274(root-specific promoters) and SEQ ID NOs:801-1019 (root-specific ORFs);SEQ ID NOs:1886-1918 (green-tissue specific promoters) and SEQ IDNOs:399-464 (green tissue-specific ORFs); SEQ ID NOs:1919-2085(panicle-specific promoters) and SEQ ID NOs:465-720 (panicle-specificpromoters); or SEQ ID NOs:2086-2143 (pollen-specific promoters) and SEQID NOs:721-800 (pollen-specific ORFs) which directs tissue-specificexpression. Thus, these nucleotide sequences exhibit promoter activityin a seed-, root-, green tissue (leaf and stem)-, panicle-, orpollen-specific manner.

Also in accordance with the present invention, nucleic acid constructsare provided that allow initiation of transcription in a“tissue-independent,” “tissue general,” or “constitutive” manner.Constructs of this embodiment invention comprise regulated transcriptioninitiation regions associated with protein translation elongation andthe compositions of this embodiment of the present invention are drawnto novel nucleotide sequences for tissue-independent, tissue-general, orconstitutive plant promoters. By “tissue-independent,” “tissue-general,”or “constitutive” is intended expression in the cells throughout a plantat most times and in most tissues. As with other promoters classified as“constitutive” (e.g., ubiquitin), some variation in absolute levels ofexpression can exist among different tissues or stages. However,constitutive promoters generally are expressed at high or moderatelevels in most, and preferably all, tissues and most, and preferablyall, developmental stages.

The present invention thus provides for isolated nucleic acid moleculescomprising a plant nucleotide sequence that directs constitutivetranscription of a linked nucleic acid fragment in a plant cell.Preferably, the nucleotide sequence is obtained or obtainable from plantgenomic DNA from a gene encoding a polypeptide which is substantiallysimilar and preferably has at least 70% amino acid sequence identity toa polypeptide encoded by an Oryza gene comprising any one of SEQ IDNOs:1598-1885 and 5960-5971, respectively (corresponding to a genecomprising an ORF comprising one of SEQ ID NOs:1-398 and 5928-5939) or afragment thereof which exhibits promoter activity in a constitutivefashion (i.e., at most times and in most tissues). Tissue-specific,i.e., seed-, root-, green tissue (leaf and stem)-, panicle-, orpollen-specific, and constitutive promoter sequences may be obtainedfrom other plant species by using the tissue-specific and constitutiveOryza promoter sequences or corresponding genes described herein asprobes to screen for homologous structural genes in other plants byhybridization under low, moderate or stringent hybridization conditions.Regions of the tissue-specific and constitutive promoter sequences ofthe present invention which are conserved among species could also beused as PCR primers to amplify a segment from a species other thanOryza, and that segment used as a hybridization probe (the latterapproach permitting higher stringency screening) or in a transcriptionassay to determine promoter activity. Moreover, the tissue-specific andconstitutive promoter sequences could be employed to identifystructurally related sequences in a database using computer algorithms.

These tissue-specific and constitutive promoters are capable of drivingthe expression of a coding sequence in a target cell, particularly in aplant cell. The promoter sequences and methods disclosed herein areuseful in regulating tissue-specific and constitutive expression,respectively, of any heterologous nucleotide sequence in a host plant inorder to vary the phenotype of that plant. These promoters can be usedwith combinations of enhancer, upstream elements, and/or activatingsequences from the 5′ flanking regions of plant expressible structuralgenes. Similarly the upstream element can be used in combination withvarious plant promoter sequences. In one embodiment the promoter andupstream element are used together to obtain at least 10-fold higherexpression of an introduced gene in monocot transgenic plants than isobtained with the maize ubiquitin 1 promoter.

In particular, all of the promoters of the invention are useful tomodify the phenotype of a plant. Various changes in the phenotype of atransgenic plant are desirable, i.e., modifying the fatty acidcomposition in a plant, altering the amino acid content of a plant,altering a plant's pathogen defense mechanism, and the like. Theseresults can be achieved by providing expression of heterologous productsor increased expression of endogenous products in plants. Alternatively,the results can be achieved by providing for a reduction of expressionof one or more endogenous products, particularly enzymes or cofactors inthe plant. These changes result in an alteration in the phenotype of thetransformed plant.

I. Definitions

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, gene refers to a nucleic acid fragment that expresses mRNAor functional RNA, or encodes a specific protein, and which includesregulatory sequences. Genes also include nonexpressed DNA segments that,for example, form recognition sequences for other proteins. Genes can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters.

The term “native” or “wild type” gene refers to a gene that is presentin the genome of an untransformed cell, i.e., a cell not having a knownmutation.

A “marker gene” encodes a selectable or screenable trait.

The term “chimeric gene” refers to any gene that contains 1) DNAsequences, including regulatory and coding sequences, that are not foundtogether in nature, or 2) sequences encoding parts of proteins notnaturally adjoined, or 3) parts of promoters that are not naturallyadjoined. Accordingly, a chimeric gene may comprise regulatory sequencesand to coding sequences that are derived from different sources, orcomprise regulatory sequences and coding sequences derived from the samesource, but arranged in a manner different from that found in nature.

A “transgene” refers to a gene that has been introduced into the genomeby transformation and is stably maintained. Transgenes may include, forexample, genes that are either heterologous or homologous to the genesof a particular plant to be transformed. Additionally, transgenes maycomprise native genes inserted into a non-native organism, or chimericgenes. The term “endogenous gene” refers to a native gene in its naturallocation in the genome of an organism. A “foreign” gene refers to a genenot normally found in the host organism but that is introduced by genetransfer.

An “oligonucleotide” corresponding to a nucleotide sequence of theinvention, e.g., for use in probing or amplification reactions, may beabout 30 or fewer nucleotides in length (e.g., 9, 12, 15, 18, 20, 21 or24, or any number between 9 and 30). Generally specific primers areupwards of 14 nucleotides in length. For optimum specificity and costeffectiveness, primers of 16 to 24 nucleotides in length may bepreferred. Those skilled in the art are well versed in the design ofprimers for use processes such as PCR. If required, probing can be donewith entire restriction fragments of the gene disclosed herein which maybe 100's or even 1000's of nucleotides in length.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

The nucleotide sequences of the invention can be introduced into anyplant. The genes to be introduced can be conveniently used in expressioncassettes for introduction and expression in any plant of interest. Suchexpression cassettes will comprise the transcriptional initiation regionof the invention linked to a nucleotide sequence of interest. Preferredpromoters include constitutive, tissue-specific, developmental-specific,inducible and/or viral promoters. Such an expression cassette isprovided with a plurality of restriction sites for insertion of the geneof interest to be under the transcriptional regulation of the regulatoryregions. The expression cassette may additionally contain selectablemarker genes. The cassette will include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region, aDNA sequence of interest, and a transcriptional and translationaltermination region functional in plants. The termination region may benative with the transcriptional initiation region, may be native withthe DNA sequence of interest, or may be derived from another source.Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau et al., 1991; Proudfoot, 1991;Sanfacon et al., 1991; Mogen et al., 1990; Munroe et al., 1990; Ballaset al., 1989; Joshi et al., 1987.

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence and excludes the non-coding sequences. Itmay constitute an “uninterrupted coding sequence”, i.e., lacking anintron, such as in a cDNA or it may include one or more introns boundedby appropriate splice junctions. An “intron” is a sequence of RNA whichis contained in the primary transcript but which is removed throughcleavage and re-ligation of the RNA within the cell to create the maturemRNA that can be translated into a protein.

The terms “open reading frame” and “ORF” refer to the amino acidsequence encoded between translation initiation and termination codonsof a coding sequence. The terms “initiation codon” and “terminationcodon” refer to a unit of three adjacent nucleotides (‘codon’) in acoding sequence that specifies initiation and chain termination,respectively, of protein synthesis (mRNA translation).

A “functional RNA” refers to an antisense RNA, ribozyme, or other RNAthat is not translated.

The term “RNA transcript” refers to the product resulting from RNApolymerase catalyzed transcription of a DNA sequence. When the RNAtranscript is a perfect complementary copy of the DNA sequence, it isreferred to as the primary transcript or it may be a RNA sequencederived from posttranscriptional processing of the primary transcriptand is referred to as the mature RNA. “Messenger RNA” (mRNA) refers tothe RNA that is without introns and that can be translated into proteinby the cell. “cDNA” refers to a single- or a double-stranded DNA that iscomplementary to and derived from mRNA.

“Regulatory sequences” and “suitable regulatory sequences” each refer tonucleotide sequences located upstream (5′ non-coding sequences), within,or downstream (3′ non-coding sequences) of a coding sequence, and whichinfluence the transcription, RNA processing or stability, or translationof the associated coding sequence. Regulatory sequences includeenhancers, promoters, translation leader sequences, introns, andpolyadenylation signal sequences. They include natural and syntheticsequences as well as sequences which may be a combination of syntheticand natural sequences. As is noted above, the term “suitable regulatorysequences” is not limited to promoters.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency(Turner et al., 1995).

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor. The use of different 3′non-coding sequences is exemplified by Ingelbrecht et al., 1989.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

“Signal peptide” refers to the amino terminal extension of apolypeptide, which is translated in conjunction with the polypeptideforming a precursor peptide and which is required for its entrance intothe secretory pathway. The term “signal sequence” refers to a nucleotidesequence that encodes the signal peptide.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which controls the expression of the coding sequence byproviding the recognition for RNA polymerase and other factors requiredfor proper transcription. “Promoter” includes a minimal promoter that isa short DNA sequence comprised of a TATA box and other sequences thatserve to specify the site of transcription initiation, to whichregulatory elements are added for control of expression. “Promoter” alsorefers to a nucleotide sequence that includes a minimal promoter plusregulatory elements that is capable of controlling the expression of acoding sequence or functional RNA. This type of promoter sequenceconsists of proximal and more distal upstream elements, the latterelements often referred to as enhancers. Accordingly, an “enhancer” is aDNA sequence which can stimulate promoter activity and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue specificity of a promoter. It is capable ofoperating in both orientations (normal or flipped), and is capable offunctioning even when moved either upstream or downstream from thepromoter. Both enhancers and other upstream promoter elements bindsequence-specific DNA-binding proteins that mediate their effects.Promoters may be derived in their entirety from a native gene, or becomposed of different elements derived from different promoters found innature, or even be comprised of synthetic DNA segments. A promoter mayalso contain DNA sequences that are involved in the binding of proteinfactors which control the effectiveness of transcription initiation inresponse to physiological or developmental conditions.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.,further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A “minimal or core promoter” thusconsists only of all basal elements needed for transcription initiation,e.g., a TATA box and/or an initiator.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter.

“Constitutive promoter” refers to a promoter that is able to express theopen reading frame (ORF) that it controls in all or nearly all of theplant tissues during all or nearly all developmental stages of theplant. Each of the transcription-activating elements do not exhibit anabsolute tissue-specificity, but mediate transcriptional activation inmost plant parts at a level of ≧1% of the level reached in the part ofthe plant in which transcription is most active.

“Regulated promoter” refers to promoters that direct gene expression notconstitutively, but in a temporally- and/or spatially-regulated manner,and includes both tissue-specific and inducible promoters. It includesnatural and synthetic sequences as well as sequences which may be acombination of synthetic and natural sequences. Different promoters maydirect the expression of a gene in different tissues or cell types, orat different stages of development, or in response to differentenvironmental conditions. New promoters of various types useful in plantcells are constantly being discovered, numerous examples may be found inthe compilation by Okamuro et al. (1989). Typical regulated promotersuseful in plants include but are not limited to safener-induciblepromoters, promoters derived from the tetracycline-inducible system,promoters derived from salicylate-inducible systems, promoters derivedfrom alcohol-inducible systems, promoters derived fromglucocorticoid-inducible system, promoters derived frompathogen-inducible systems, and promoters derived fromecdysome-inducible systems.

“Tissue-specific promoter” refers to regulated promoters that are notexpressed in all plant cells but only in one or more cell types inspecific organs (such as leaves or seeds), specific tissues (such asembryo or cotyledon), or specific cell types (such as leaf parenchyma orseed storage cells). These also include promoters that are temporallyregulated, such as in early or late embryogenesis, during fruit ripeningin developing seeds or fruit, in fully differentiated leaf, or at theonset of senescence.

“Inducible promoter” refers to those regulated promoters that can beturned on in one or more cell types by an external stimulus, such as achemical, light, hormone, stress, or a pathogen.

“Operably-linked” refers to the association of nucleic acid sequences onsingle nucleic acid fragment so that the function of one is affected bythe other. For example, a regulatory DNA sequence is said to be“operably linked to” or “associated with” a DNA sequence that codes foran RNA or a polypeptide if the two sequences are situated such that theregulatory DNA sequence affects expression of the coding DNA sequence(i.e., that the coding sequence or functional RNA is under thetranscriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

“Expression” refers to the transcription and/or translation of anendogenous gene, ORF or portion thereof, or a transgene in plants. Forexample, in the case of antisense constructs, expression may refer tothe transcription of the antisense DNA only. In addition, expressionrefers to the transcription and stable accumulation of sense (mRNA) orfunctional RNA. Expression may also refer to the production of protein.

“Specific expression” is the expression of gene products which islimited to one or a few plant tissues (spatial limitation) and/or to oneor a few plant developmental stages (temporal limitation). It isacknowledged that hardly a true specificity exists: promoters seem to bepreferably switch on in some tissues, while in other tissues there canbe no or only little activity. This phenomenon is known as leakyexpression. However, with specific expression in this invention is meantpreferable expression in one or a few plant tissues.

The “expression pattern” of a promoter (with or without enhancer) is thepattern of expression levels which shows where in the plant and in whatdevelopmental stage transcription is initiated by said promoter.Expression patterns of a set of promoters are said to be complementarywhen the expression pattern of one promoter shows little overlap withthe expression pattern of the other promoter. The level of expression ofa promoter can be determined by measuring the ‘steady state’concentration of a standard transcribed reporter mRNA. This measurementis indirect since the concentration of the reporter mRNA is dependentnot only on its synthesis rate, but also on the rate with which the mRNAis degraded. Therefore, the steady state level is the product ofsynthesis rates and degradation rates.

The rate of degradation can however be considered to proceed at a fixedrate when the transcribed sequences are identical, and thus this valuecan serve as a measure of synthesis rates. When promoters are comparedin this way techniques available to those skilled in the art arehybridization S1-RNAse analysis, northern blots and competitive RT-PCR.This list of techniques in no way represents all available techniques,but rather describes commonly used procedures used to analyzetranscription activity and expression levels of mRNA.

The analysis of transcription start points in practically all promotershas revealed that there is usually no single base at which transcriptionstarts, but rather a more or less clustered set of initiation sites,each of which accounts for some start points of the mRNA. Since thisdistribution varies from promoter to promoter the sequences of thereporter mRNA in each of the populations would differ from each other.Since each mRNA species is more or less prone to degradation, no singledegradation rate can be expected for different reporter mRNAs. It hasbeen shown for various eukaryotic promoter sequences that the sequencesurrounding the initiation site (‘initiator’) plays an important role indetermining the level of RNA expression directed by that specificpromoter. This includes also part of the transcribed sequences. Thedirect fusion of promoter to reporter sequences would therefore lead tosuboptimal levels of transcription.

A commonly used procedure to analyze expression patterns and levels isthrough determination of the ‘steady state’ level of proteinaccumulation in a cell. Commonly used candidates for the reporter gene,known to those skilled in the art are beta-glucuronidase (GUS),chloramphenicol acetyl transferase (CAT) and proteins with fluorescentproperties, such as green fluorescent protein (GFP) from Aequoravictoria. In principle, however, many more proteins are suitable forthis purpose, provided the protein does not interfere with essentialplant functions. For quantification and determination of localization anumber of tools are suited. Detection systems can readily be created orare available which are based on, e.g., immunochemical, enzymatic,fluorescent detection and quantification. Protein levels can bedetermined in plant tissue extracts or in intact tissue using in situanalysis of protein expression.

Generally, individual transformed lines with one chimeric promoterreporter construct will vary in their levels of expression of thereporter gene. Also frequently observed is the phenomenon that suchtransformants do not express any detectable product (RNA or protein).The variability in expression is commonly ascribed to ‘positioneffects’, although the molecular mechanisms underlying this inactivityare usually not clear.

“Overexpression” refers to the level of expression in transgenic cellsor organisms that exceeds levels of expression in normal oruntransformed (nontransgenic) cells or organisms.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of protein from anendogenous gene or a transgene.

“Gene silencing” refers to homology-dependent suppression of viralgenes, transgenes, or endogenous nuclear genes. Gene silencing may betranscriptional, when the suppression is due to decreased transcriptionof the affected genes, or post-transcriptional, when the suppression isdue to increased turnover (degradation) of RNA species homologous to theaffected genes (English et al., 1996). Gene silencing includesvirus-induced gene silencing (Ruiz et al. 1998).

The terms “heterologous DNA sequence,” “exogenous DNA segment” or“heterologous nucleic acid,” as used herein, each refer to a sequencethat originates from a source foreign to the particular host cell or, iffrom the same source, is modified from its original form. Thus, aheterologous gene in a host cell includes a gene that is endogenous tothe particular host cell but has been modified through, for example, theuse of DNA shuffling. The terms also include non-naturally occurringmultiple copies of a naturally occurring DNA sequence. Thus, the termsrefer to a DNA segment that is foreign or heterologous to the cell, orhomologous to the cell but in a position within the host cell nucleicacid in which the element is not ordinarily found. Exogenous DNAsegments are expressed to yield exogenous polypeptides. A “homologous”DNA sequence is a DNA sequence that is naturally associated with a hostcell into which it is introduced.

“Homologous to” in the context of nucleotide sequence identity refers tothe similarity between the nucleotide sequence of two nucleic acidmolecules or between the amino acid sequences of two protein molecules.Estimates of such homology are provided by either DNA-DNA or DNA-RNAhybridization under conditions of stringency as is well understood bythose skilled in the art (as described in Haines and Higgins (eds.),Nucleic Acid Hybridization, IRL Press, Oxford, U.K.), or by thecomparison of sequence similarity between two nucleic acids or proteins.

The term “substantially similar” refers to nucleotide and amino acidsequences that represent functional and/or structural equivalents ofOryza sequences disclosed herein.

In its broadest sense, the term “substantially similar” when used hereinwith respect to a nucleotide sequence means that the nucleotide sequenceis part of a gene which encodes a polypeptide having substantially thesame structure and function as a polypeptide encoded by a gene for thereference nucleotide sequence, e.g., the nucleotide sequence comprises apromoter from a gene that is the ortholog of the gene corresponding tothe reference nucleotide sequence, as well as promoter sequences thatare structurally related the promoter sequences particularly exemplifiedherein, i.e., the substantially similar promoter sequences hybridize tothe complement of the promoter sequences exemplified herein under highor very high stringency conditions. For example, altered nucleotidesequences which simply reflect the degeneracy of the genetic code butnonetheless encode amino acid sequences that are identical to aparticular amino acid sequence are substantially similar to theparticular sequences. The term “substantially similar” also includesnucleotide sequences wherein the sequence has been modified, forexample, to optimize expression in particular cells, as well asnucleotide sequences encoding a variant polypeptide having one or moreamino acid substitutions relative to the (unmodified) polypeptideencoded by the reference sequence, which substitution(s) does not alterthe activity of the variant polypeptide relative to the unmodifiedpolypeptide.

In its broadest sense, the term “substantially similar” when used hereinwith respect to polypeptide means that the polypeptide has substantiallythe same structure and function as the reference polypeptide. Inaddition, amino acid sequences that are substantially similar to aparticular sequence are those wherein overall amino acid identity is atleast 65% or greater to the instant sequences. Modifications that resultin equivalent nucleotide or amino acid sequences are well within theroutine skill in the art. The percentage of amino acid sequence identitybetween the substantially similar and the reference polypeptide is atleast 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, andeven 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, up to atleast 99%, wherein the reference polypeptide is an Oryza polypeptideencoded by a gene with a promoter having any one of SEQ ID NOs:1-350 and1051-1551, e.g., a nucleotide sequence comprising an open reading framehaving any one of SEQ ID NOs:351-700 or 1552-2052 which encodes one ofSEQ ID Nos:701-1050 or 2053-2553. One indication that two polypeptidesare substantially similar to each other, besides having substantiallythe same function, is that an agent, e.g., an antibody, whichspecifically binds to one of the polypeptides, specifically binds to theother.

Sequence comparisons maybe carried out using a Smith-Waterman sequencealignment algorithm (see e.g., Waterman (1995)). The localS program,version 1.16, is preferably used with following parameters: match: 1,mismatch penalty: 0.33, open-gap penalty: 2, extended-gap penalty: 2.

Moreover, a nucleotide sequence that is “substantially similar” to areference nucleotide sequence is said to be “equivalent” to thereference nucleotide sequence. The skilled artisan recognizes thatequivalent nucleotide sequences encompassed by this invention can alsobe defined by their ability to hybridize, under low, moderate and/orstringent conditions (e.g., 0.1×SSC, 0.1% SDS, 65° C.), with thenucleotide sequences that are within the literal scope of the instantclaims.

What is meant by “substantially the same activity” when used inreference to a polynucleotide or polypeptide fragment is that thefragment has at least 65%, 66%, 67%, 68%, 69%, 70%, e.g., 71%, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%,88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%,98%, up to at least 99% of the activity of the full lengthpolynucleotide or full length polypeptide.

“Target gene” refers to a gene on the replicon that expresses thedesired target coding sequence, functional RNA, or protein. The targetgene is not essential for replicon replication. Additionally, targetgenes may comprise native non-viral genes inserted into a non-nativeorganism, or chimeric genes, and will be under the control of suitableregulatory sequences. Thus, the regulatory sequences in the target genemay come from any source, including the virus. Target genes may includecoding sequences that are either heterologous or homologous to the genesof a particular plant to be transformed. However, target genes do notinclude native viral genes. Typical target genes include, but are notlimited to genes encoding a structural protein, a seed storage protein,a protein that conveys herbicide resistance, and a protein that conveysinsect resistance. Proteins encoded by target genes are known as“foreign proteins”. The expression of a target gene in a plant willtypically produce an altered plant trait.

The term “altered plant trait” means any phenotypic or genotypic changein a transgenic plant relative to the wild-type or non-transgenic planthost.

“Replication gene” refers to a gene encoding a viral replicationprotein. In addition to the ORF of the replication protein, thereplication gene may also contain other overlapping or non-overlappingORF(s), as are found in viral sequences in nature. While not essentialfor replication, these additional ORFs may enhance replication and/orviral DNA accumulation. Examples of such additional ORFs are AC3 and AL3in ACMV and TGMV geminiviruses, respectively.

“Chimeric trans-acting replication gene” refers either to a replicationgene in which the coding sequence of a replication protein is under thecontrol of a regulated plant promoter other than that in the nativeviral replication gene, or a modified native viral replication gene, forexample, in which a site specific sequence(s) is inserted in the 5′transcribed but untranslated region. Such chimeric genes also includeinsertion of the known sites of replication protein binding between thepromoter and the transcription start site that attenuate transcriptionof viral replication protein gene.

“Chromosomally-integrated” refers to the integration of a foreign geneor DNA construct into the host DNA by covalent bonds. Where genes arenot “chromosomally integrated” they may be “transiently expressed.”Transient expression of a gene refers to the expression of a gene thatis not integrated into the host chromosome but functions independently,either as part of an autonomously replicating plasmid or expressioncassette, for example, or as part of another biological system such as avirus.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. Host cells containing the transformed nucleic acidfragments are referred to as “transgenic” cells, and organismscomprising transgenic cells are referred to as “transgenic organisms”.Examples of methods of transformation of plants and plant cells includeAgrobacterium-mediated transformation (De Blaere et al., 1987) andparticle bombardment technology (Klein et al. 1987; U.S. Pat. No.4,945,050). Whole plants may be regenerated from transgenic cells bymethods well known to the skilled artisan (see, for example, Fromm etal., 1990).

“Transformed,” “transgenic,” and “recombinant” refer to a host organismsuch as a bacterium or a plant into which a heterologous nucleic acidmolecule has been introduced. The nucleic acid molecule can be stablyintegrated into the genome generally known in the art and are disclosedin Sambrook et al., 1989. See also Innis et al., 1995 and Gelfand, 1995;and Innis and Gelfand, 1999. Known methods of PCR include, but are notlimited to, methods using paired primers, nested primers, singlespecific primers, degenerate primers, gene-specific primers,vector-specific primers, partially mismatched primers, and the like. Forexample, “transformed,” “transformant,” and “transgenic” plants or callihave been through the transformation process and contain a foreign geneintegrated into their chromosome. The term “untransformed” refers tonormal plants that have not been through the transformation process.

“Transiently transformed” refers to cells in which transgenes andforeign DNA have been introduced (for example, by such methods asAgrobacterium-mediated transformation or biolistic bombardment), but notselected for stable maintenance.

“Stably transformed” refers to cells that have been selected andregenerated on a selection media following transformation.

“Transient expression” refers to expression in cells in which a virus ora transgene is introduced by viral infection or by such methods asAgrobacterium-mediated transformation, electroporation, or biolisticbombardment, but not selected for its stable maintenance.

“Genetically stable” and “heritable” refer to chromosomally-integratedgenetic elements that are stably maintained in the plant and stablyinherited by progeny through successive generations.

“Primary transformant” and “TO generation” refer to transgenic plantsthat are of the same genetic generation as the tissue which wasinitially transformed (i.e., not having gone through meiosis andfertilization since transformation).

“Secondary transformants” and the “T1, T2, T3, etc. generations” referto transgenic plants derived from primary transformants through one ormore meiotic and fertilization cycles. They may be derived byself-fertilization of primary or secondary transformants or crosses ofprimary or secondary transformants with other transformed oruntransformed plants.

“Wild-type” refers to a virus or organism found in nature without anyknown mutation.

“Genome” refers to the complete genetic material of an organism.

The term “nucleic acid” refers to deoxyribonucleotides orribonucleotides and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base which is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides which have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also implicitlyencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues (Batzer et al., 1991; Ohtsuka et al., 1985;Rossolini et al. 1994). A “nucleic acid fragment” is a fraction of agiven nucleic acid molecule. In higher plants, deoxyribonucleic acid(DNA) is the genetic material while ribonucleic acid (RNA) is involvedin the transfer of information contained within DNA into proteins. Theterm “nucleotide sequence” refers to a polymer of DNA or RNA which canbe single- or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases capable of incorporation intoDNA or RNA polymers. The terms “nucleic acid” or “nucleic acid sequence”may also be used interchangeably with gene, cDNA, DNA and RNA encoded bya gene.

The invention encompasses isolated or substantially purified nucleicacid or protein compositions. In the context of the present invention,an “isolated” or “purified” DNA molecule or an “isolated” or “purified”polypeptide is a DNA molecule or polypeptide that, by the hand of man,exists apart from its native environment and is therefore not a productof nature. An isolated DNA molecule or polypeptide may exist in apurified form or may exist in a non-native environment such as, forexample, a transgenic host cell. For example, an “isolated” or“purified” nucleic acid molecule or protein, or biologically activeportion thereof, is substantially free of other cellular material, orculture medium when produced by recombinant techniques, or substantiallyfree of chemical precursors or other chemicals when chemicallysynthesized. Preferably, an “isolated” nucleic acid is free of sequences(preferably protein encoding sequences) that naturally flank the nucleicacid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleicacid molecule in genomic DNA of the cell from which the nucleic acid isderived. A protein that is substantially free of cellular materialincludes preparations of protein or polypeptide having less than about30%, 20%, 10%, 5%, (by dry weight) of contaminating protein. When theprotein of the invention, or biologically active portion thereof, isrecombinantly produced, preferably culture medium represents less thanabout 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors ornon-protein of interest chemicals.

The nucleotide sequences of the invention include both the naturallyoccurring sequences as well as mutant (variant) forms. Such variantswill continue to possess the desired activity, i.e., either promoteractivity or the activity of the product encoded by the open readingframe of the non-variant nucleotide sequence.

Thus, by “variants” is intended substantially similar sequences. Fornucleotide sequences comprising an open reading frame, variants includethose sequences that, because of the degeneracy of the genetic code,encode the identical amino acid sequence of the native protein.Naturally occurring allelic variants such as these can be identifiedwith the use of well-known molecular biology techniques, as, forexample, with polymerase chain reaction (PCR) and hybridizationtechniques. Variant nucleotide sequences also include syntheticallyderived nucleotide sequences, such as those generated, for example, byusing site-directed mutagenesis and for open reading frames, encode thenative protein, as well as those that encode a polypeptide having aminoacid substitutions relative to the native protein. Generally, nucleotidesequence variants of the invention will have at least 40, 50, 60, to70%, e.g., preferably 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98% and 99%nucleotide sequence identity to the native (wild type or endogenous)nucleotide sequence.

“Conservatively modified variations” of a particular nucleic acidsequence refers to those nucleic acid sequences that encode identical oressentially identical amino acid sequences, or where the nucleic acidsequence does not encode an amino acid sequence, to essentiallyidentical sequences. Because of the degeneracy of the genetic code, alarge number of functionally identical nucleic acids encode any givenpolypeptide. For instance the codons CGT, CGC, CGA, CGG, AGA, and AGGall encode the amino acid arginine. Thus, at every position where anarginine is specified by a codon, the codon can be altered to any of thecorresponding codons described without altering the encoded protein.Such nucleic acid variations are “silent variations” which are onespecies of “conservatively modified variations.” Every nucleic acidsequence described herein which encodes a polypeptide also describesevery possible silent variation, except where otherwise noted. One ofskill will recognize that each codon in a nucleic acid (except ATG,which is ordinarily the only codon for methionine) can be modified toyield a functionally identical molecule by standard techniques.Accordingly, each “silent variation” of a nucleic acid which encodes apolypeptide is implicit in each described sequence.

The nucleic acid molecules of the invention can be “optimized” forenhanced expression in plants of interest. See, for example, EPA 035472;WO 91/16432; Perlak et al., 1991; and Murray et al., 1989. In thismanner, the open reading frames in genes or gene fragments can besynthesized utilizing plant-preferred codons. See, for example, Campbelland Gowri, 1990 for a discussion of host-preferred codon usage. Thus,the nucleotide sequences can be optimized for expression in any plant.It is recognized that all or any part of the gene sequence may beoptimized or synthetic. That is, synthetic or partially optimizedsequences may also be used. Variant nucleotide sequences and proteinsalso encompass sequences and protein derived from a mutagenic andrecombinogenic procedure such as DNA shuffling. With such a procedure,one or more different coding sequences can be manipulated to create anew polypeptide possessing the desired properties. In this manner,libraries of recombinant polynucleotides are generated from a populationof related sequence polynucleotides comprising sequence regions thathave substantial sequence identity and can be homologously recombined invitro or in vivo. Strategies for such DNA shuffling are known in theart. See, for example, Stemmer, 1994; Stemmer, 1994; Crameri et al.,1997; Moore et al., 1997; Zhang et al., 1997; Crameri et al., 1998; andU.S. Pat. Nos. 5,605,793 and 5,837,458.

By “variant” polypeptide is intended a polypeptide derived from thenative protein by deletion (so-called truncation) or addition of one ormore amino acids to the N-terminal and/or C-terminal end of the nativeprotein; deletion or addition of one or more amino acids at one or moresites in the native protein; or substitution of one or more amino acidsat one or more sites in the native protein. Such variants may resultfrom, for example, genetic polymorphism or from human manipulation.Methods for such manipulations are generally known in the art.

Thus, the polypeptides may be altered in various ways including aminoacid substitutions, deletions, truncations, and insertions. Methods forsuch manipulations are generally known in the art. For example, aminoacid sequence variants of the polypeptides can be prepared by mutationsin the DNA. Methods for mutagenesis and nucleotide sequence alterationsare well known in the art. See, for example, Kunkel, 1985; Kunkel etal., 1987; U.S. Pat. No. 4,873,192; Walker and Gaastra, 1983 and thereferences cited therein. Guidance as to appropriate amino acidsubstitutions that do not affect biological activity of the protein ofinterest may be found in the model of Dayhoff et al. (1978).Conservative substitutions, such as exchanging one amino acid withanother having similar properties, are preferred.

Individual substitutions deletions or additions that alter, add ordelete a single amino acid or a small percentage of amino acids(typically less than 5%, more typically less than 1%) in an encodedsequence are “conservatively modified variations,” where the alterationsresult in the substitution of an amino acid with a chemically similaramino acid. Conservative substitution tables providing functionallysimilar amino acids are well known in the art. The following five groupseach contain amino acids that are conservative substitutions for oneanother: Aliphatic: Glycine (G), Alanine (A), Valine (V), Leucine (L),Isoleucine (I); Aromatic: Phenylalanine (F), Tyrosine (Y), Tryptophan(W); Sulfur-containing: Methionine (M), Cysteine (C); Basic: Arginine I,Lysine (K), Histidine (H); Acidic: Aspartic acid (D), Glutamic acid (E),Asparagine (N), Glutamine (Q). See also, Creighton, 1984. In addition,individual substitutions, deletions or additions which alter, add ordelete a single amino acid or a small percentage of amino acids in anencoded sequence are also “conservatively modified variations.”

“Expression cassette” as used herein means a DNA sequence capable ofdirecting expression of a particular nucleotide sequence in anappropriate host cell, comprising a promoter operably linked to thenucleotide sequence of interest which is operably linked to terminationsignals. It also typically comprises sequences required for propertranslation of the nucleotide sequence. The coding region usually codesfor a protein of interest but may also code for a functional RNA ofinterest, for example antisense RNA or a nontranslated RNA, in the senseor antisense direction. The expression cassette comprising thenucleotide sequence of interest may be chimeric, meaning that at leastone of its components is heterologous with respect to at least one ofits other components. The expression cassette may also be one which isnaturally occurring but has been obtained in a recombinant form usefulfor heterologous expression. The expression of the nucleotide sequencein the expression cassette may be under the control of a constitutivepromoter or of an inducible promoter which initiates transcription onlywhen the host cell is exposed to some particular external stimulus. Inthe case of a multicellular organism, the promoter can also be specificto a particular tissue or organ or stage of development.

“Vector” is defined to include, inter alia, any plasmid, cosmid, phageor Agrobacterium binary vector in double or single stranded linear orcircular form which may or may not be self transmissible or mobilizable,and which can transform prokaryotic or eukaryotic host either byintegration into the cellular genome or exist extrachromosomally (e.g.autonomous replicating plasmid with an origin of replication).

Specifically included are shuttle vectors by which is meant a DNAvehicle capable, naturally or by design, of replication in two differenthost organisms, which may be selected from actinomycetes and relatedspecies, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast orfungal cells).

Preferably the nucleic acid in the vector is under the control of, andoperably linked to, an appropriate promoter or other regulatory elementsfor transcription in a host cell such as a microbial, e.g. bacterial, orplant cell. The vector may be a bi-functional expression vector whichfunctions in multiple hosts. In the case of genomic DNA, this maycontain its own promoter or other regulatory elements and in the case ofcDNA this may be under the control of an appropriate promoter or otherregulatory elements for expression in the host cell.

“Cloning vectors” typically contain one or a small number of restrictionendonuclease recognition sites at which foreign DNA sequences can beinserted in a determinable fashion without loss of essential biologicalfunction of the vector, as well as a marker gene that is suitable foruse in the identification and selection of cells transformed with thecloning vector. Marker genes typically include genes that providetetracycline resistance, hygromycin resistance or ampicillin resistance.

A “transgenic plant” is a plant having one or more plant cells thatcontain an expression vector.

“Plant tissue” includes differentiated and undifferentiated tissues orplants, including but not limited to roots, stems, shoots, leaves,pollen, seeds, tumor tissue and various forms of cells and culture suchas single cells, protoplast, embryos, and callus tissue. The planttissue may be in plants or in organ, tissue or cell culture.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence”, (b) “comparison window”, (c) “sequence identity”, (d)“percentage of sequence identity”, and (e) “substantial identity”.

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm. Preferred,non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller, 1988; the local homology algorithm of Smith et al.1981; the homology alignment algorithm of Needleman and Wunsch 1970; thesearch-for-similarity-method of Pearson and Lipman 1988; the algorithmof Karlin and Altschul, 1990, modified as in Karlin and Altschul, 1993.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.1988; Higgins et al. 1989; Corpet et al. 1988; Huang et al. 1992; andPearson et al. 1994. The ALIGN program is based on the algorithm ofMyers and Miller, supra. The BLAST programs of Altschul et al., 1990,are based on the algorithm of Karlin and Altschul supra.

Software for performing BLAST analyses is publicly available through thewebsite of the National Center for Biotechnology Information (NCBI).This algorithm involves first identifying high scoring sequence pairs(HSPs) by identifying short words of length W in the query sequence,which either match or satisfy some positive-valued threshold score Twhen aligned with a word of the same length in a database sequence. T isreferred to as the neighborhood word score threshold (Altschul et al.,1990). These initial neighborhood word hits act as seeds for initiatingsearches to find longer HSPs containing them. The word hits are thenextended in both directions along each sequence for as far as thecumulative alignment score can be increased. Cumulative scores arecalculated using, for nucleotide sequences, the parameters M (rewardscore for a pair of matching residues; always >0) and N (penalty scorefor mismatching residues; always <0). For amino acid sequences, ascoring matrix is used to calculate the cumulative score. Extension ofthe word hits in each direction are halted when the cumulative alignmentscore falls off by the quantity X from its maximum achieved value, thecumulative score goes to zero or below due to the accumulation of one ormore negative-scoring residue alignments, or the end of either sequenceis reached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences (see, e.g., Karlin & Altschul (1993). One measure ofsimilarity provided by the BLAST algorithm is the smallest sumprobability (P(N)), which provides an indication of the probability bywhich a match between two nucleotide or amino acid sequences would occurby chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleic acid sequence to the reference nucleicacid sequence is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. 1997.Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform aniterated search that detects distant relationships between molecules.See Altschul et al., supra. When utilizing BLAST, Gapped BLAST,PSI-BLAST, the default parameters of the respective programs (e.g.BLASTN for nucleotide sequences, BLASTX for proteins) can be used. TheBLASTN program (for nucleotide sequences) uses as defaults a wordlength(W) of 11, an expectation (E) of 10, a cutoff of 100, M=5, N=−4, and acomparison of both strands. For amino acid sequences, the BLASTP programuses as defaults a wordlength (W) of 3, an expectation (E) of 10, andthe BLOSUM62 scoring matrix (see Henikoff & Henikoff, 1989). See thewebsite of the NCBI. Alignment may also be performed manually byinspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide or amino acid residue matches and anidentical percent sequence identity when compared to the correspondingalignment generated by the preferred program.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid or polypeptide sequences makes reference to theresidues in the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity) and therefore do not change the functional propertiesof the molecule. When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity.” Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e)(i) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%,91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%,or 99% sequence identity, compared to a reference sequence using one ofthe alignment programs described using standard parameters. One of skillin the art will recognize that these values can be appropriatelyadjusted to determine corresponding identity of proteins encoded by twonucleotide sequences by taking into account codon degeneracy, amino acidsimilarity, reading frame positioning, and the like. Substantialidentity of amino acid sequences for these purposes normally meanssequence identity of at least 70%, more preferably at least 80%, 90%,and most preferably at least 95%.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions(see below). Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T_(m)) for the specificsequence at a defined ionic strength and pH. However, stringentconditions encompass temperatures in the range of about 1° C. to about20° C., depending upon the desired degree of stringency as otherwisequalified herein. Nucleic acids that do not hybridize to each otherunder stringent conditions are still substantially identical if thepolypeptides they encode are substantially identical. This may occur,e.g., when a copy of a nucleic acid is created using the maximum codondegeneracy permitted by the genetic code. One indication that twonucleic acid sequences are substantially identical is when thepolypeptide encoded by the first nucleic acid is immunologically crossreactive with the polypeptide encoded by the second nucleic acid.

(e)(ii) The term “substantial identity” in the context of a peptideindicates that a peptide comprises a sequence with at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%, 91%,92%, 93%, or 94%, or even more preferably, 95%, 96%, 97%, 98% or 99%,sequence identity to the reference sequence over a specified comparisonwindow. Preferably, optimal alignment is conducted using the homologyalignment algorithm of Needleman and Wunsch (1970). An indication thattwo peptide sequences are substantially identical is that one peptide isimmunologically reactive with antibodies raised against the secondpeptide. Thus, a peptide is substantially identical to a second peptide,for example, where the two peptides differ only by a conservativesubstitution.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted above, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridization are sequence dependent, andare different under different environmental parameters. The T_(m) is thetemperature (under defined ionic strength and pH) at which 50% of thetarget sequence hybridizes to a perfectly matched probe. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the T_(m) can be approximated from theequation of Meinkoth and Wahl, 1984; T_(m) 81.5° C.+16.6 (log M)+0.41 (%GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% form is the percentage of formamide in the hybridization solution, andL is the length of the hybrid in base pairs. T_(m) is reduced by about1° C. for each 1% of mismatching; thus, T_(m), hybridization, and/orwash conditions can be adjusted to hybridize to sequences of the desiredidentity. For example, if sequences with >90% identity are sought, theT_(m) can be decreased 10° C. Generally, stringent conditions areselected to be about 5° C. lower than the thermal melting point I forthe specific sequence and its complement at a defined ionic strength andpH. However, severely stringent conditions can utilize a hybridizationand/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point I;moderately stringent conditions can utilize a hybridization and/or washat 6, 7, 8, 9, or 10° C. lower than the thermal melting point I; lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point I. Using theequation, hybridization and wash compositions, and desired T, those ofordinary skill will understand that variations in the stringency ofhybridization and/or wash solutions are inherently described. If thedesired degree of mismatching results in a T of less than 45° C.(aqueous solution) or 32° C. (formamide solution), it is preferred toincrease the SSC concentration so that a higher temperature can be used.An extensive guide to the hybridization of nucleic acids is found inTijssen, 1993. Generally, highly stringent hybridization and washconditions are selected to be about 5° C. lower than the thermal meltingpoint T_(m) for the specific sequence at a defined ionic strength andpH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook, infra, for adescription of SSC buffer). Often, a high stringency wash is preceded bya low stringency wash to remove background probe signal. An examplemedium stringency wash for a duplex of, e.g., more than 100 nucleotides,is 1×SSC at 45° C. for 15 minutes. An example low stringency wash for aduplex of, e.g., more than 100 nucleotides, is 4-6×SSC at 40° C. for 15minutes. For short probes (e.g., about 10 to 50 nucleotides), stringentconditions typically involve salt concentrations of less than about 1.5M, more preferably about 0.01 to 1.0 M, Na ion concentration (or othersalts) at pH 7.0 to 8.3, and the temperature is typically at least about30° C. and at least about 60° C. for long robes (e.g., >50 nucleotides).Stringent conditions may also be achieved with the addition ofdestabilizing agents such as formamide. In general, a signal to noiseratio of 2× (or higher) than that observed for an unrelated probe in theparticular hybridization assay indicates detection of a specifichybridization. Nucleic acids that do not hybridize to each other understringent conditions are still substantially identical if the proteinsthat they encode are substantially identical. This occurs, e.g., when acopy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1 MNaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

The following are examples of sets of hybridization/wash conditions thatmay be used to clone orthologous nucleotide sequences that aresubstantially identical to reference nucleotide sequences of the presentinvention: a reference nucleotide sequence preferably hybridizes to thereference nucleotide sequence in 7% sodium dodecyl sulfate (SDS), 0.5 MNaPO₄, 1 mM EDTA at 50° C. with washing in 2×SSC, 0.1% SDS at 50° C.,more desirably in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mMEDTA at 50° C. with washing in 1×SSC, 0.1% SDS at 50° C., more desirablystill in 7% sodium dodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50°C. with washing in 0.5×SSC, 0.1% SDS at 50° C., preferably in 7% sodiumdodecyl sulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in0.1×SSC, 0.1% SDS at 50° C., more preferably in 7% sodium dodecylsulfate (SDS), 0.5 M NaPO₄, 1 mM EDTA at 50° C. with washing in 0.1×SSC,0.1% SDS at 65° C.

“DNA shuffling” is a method to introduce mutations or rearrangements,preferably randomly, in a DNA molecule or to generate exchanges of DNAsequences between two or more DNA molecules, preferably randomly. TheDNA molecule resulting from DNA shuffling is a shuffled DNA moleculethat is a non-naturally occurring DNA molecule derived from at least onetemplate DNA molecule. The shuffled DNA preferably encodes a variantpolypeptide modified with respect to the polypeptide encoded by thetemplate DNA, and may have an altered biological activity with respectto the polypeptide encoded by the template DNA.

“Recombinant DNA molecule’ is a combination of DNA sequences that arejoined together using recombinant DNA technology and procedures used tojoin together DNA sequences as described, for example, in Sambrook etal., 1989.

The word “plant” refers to any plant, particularly to seed plant, and“plant cell” is a structural and physiological unit of the plant, whichcomprises a cell wall but may also refer to a protoplast. The plant cellmay be in form of an isolated single cell or a cultured cell, or as apart of higher organized unit such as, for example, a plant tissue, or aplant organ.

“Significant increase” is an increase that is larger than the margin oferror inherent in the measurement technique, preferably an increase byabout 2-fold or greater.

“Significantly less” means that the decrease is larger than the marginof error inherent in the measurement technique, preferably a decrease byabout 2-fold or greater.

II. Nucleic Acid Molecules of the Invention

The invention relates to an isolated plant, e.g., Oryza, nucleic acidmolecule which directs the expression of linked nucleic acid segment ina plant, e.g., in a particular tissue or constitutively, as well as thecorresponding open reading frame and encoded product. The nucleic acidmolecule, e.g., one which comprises a promoter, can be used tooverexpress a linked nucleic acid segment so as to express a product ina constitutive, tissue-specific or tissue-preferential manner, or toalter the expression of the product, e.g., via the use of antisensevectors or by “knocking out” the expression of at least one genomic copyof the gene.

The nucleic acid molecules of the invention can be obtained or isolatedfrom any plant or non-plant source, or produced synthetically by purleychemical means. Preferred sources include, but are not limited to, corn(Zea mays), Brassica sp. (e.g., B. napus, B. rapa, B. juncea),particularly those Brassica species useful as sources of seed oil,alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale),sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet(Pennisetum glaucum), proso millet (Panicum miliaceum), foxtail millet(Setaria italica), finger millet (Eleusine coracana)), sunflower(Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticumaestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato(Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypiumbarbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava(Manihot esculenta), coffee (Cofea spp.), coconut (Cocos nucifera),pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobromacacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Perseaultilane), fig (Ficus casica), guava (Psidium guajava), mango (Mangiferaindica), olive (Olea europaea), papaya (Carica papaya), cashew(Anacardium occidentale), macadamia (Macadamia integrifolia), almond(Prunus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharumspp.), oats, duckweed (Lemna), barley, vegetables, ornamentals, andconifers.

Duckweed (Lemna, see WO 00/07210) includes members of the familyLemnaceae. There are known four genera and 34 species of duckweed asfollows: genus Lemna (L. aequinoctialis, L. disperma, L. ecuadoriensis,L. gibba, L. japonica, L. minor, L. miniscula,

L. obscura, L. perpusilla, L. tenera, L. trisulca, L. turionifera, L.valdiviana); genus Spirodela (S. intermedia, S. polyrrhiza, S.punctata); genus Woffia (Wa. Angusta, Wa. Arrhiza, Wa. Australina, Wa.Borealis, Wa. Brasiliensis, Wa. Columbiana, Wa. Elongata, Wa. Globosa,Wa. Microscopica, Wa. Neglecta) and genus Wofiella (Wl. ultila, Wl.ultilane n, Wl. gladiata, Wl. ultila, Wl. lingulata, Wl. repunda, Wl.rotunda, and Wl. neotropica). Any other genera or species of Lemnaceae,if they exist, are also aspects of the present invention. Lemna gibba,Lemna minor, and Lemna miniscula are preferred, with Lemna minor andLemna miniscula being most preferred. Lemna species can be classifiedusing the taxonomic scheme described by Landolt, BiosystematicInvestigation on the Family of Duckweeds: The family of Lemnaceae—AMonograph Study. Geobatanischen Institut ETH, Stiftung Rubel, Zurich(1986)).

Vegetables from which to obtain or isolate the nucleic acid molecules ofthe invention include, but are not limited to, tomatoes (Lycopersiconesculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolusvulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), andmembers of the genus Cucumis such as cucumber (C. sativus), cantaloupe(C. cantalupensis), and musk melon (C. melo). Ornamentals from which toobtain or isolate the nucleic acid molecules of the invention include,but are not limited to, azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the present invention include, for example, pinessuch as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), andMonterey pine (Pinus radiata), Douglas-fir (Pseudotsuga menziesii);Western hemlock (Tsuga ultilane); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Leguminous plants from which the nucleic acid molecules of the inventioncan be isolated or obtained include, but are not limited to, beans andpeas. Beans include guar, locust bean, fenugreek, soybean, garden beans,cowpea, mungbean, lima bean, fava bean, lentils, chickpea, and the like.Legumes include, but are not limited to, Arachis, e.g., peanuts, Vicia,e.g., crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea,Lupinus, e.g., lupine, trifolium, Phaseolus, e.g., common bean and limabean, Pisum, e.g., field bean, Melilotus, e.g., clover, Medicago, e.g.,alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, and false indigo.Preferred forage and turf grass from which the nucleic acid molecules ofthe invention can be isolated or obtained for use in the methods of theinvention include, but are not limited to, alfalfa, orchard grass, tallfescue, perennial ryegrass, creeping bent grass, and redtop.

Other preferred sources of the nucleic acid molecules of the inventioninclude Acacia, aneth, artichoke, arugula, blackberry, canola, cilantro,clementines, escarole, eucalyptus, fennel, grapefruit, honey dew,jicama, kiwifruit, lemon, lime, mushroom, nut, okra, orange, parsley,persimmon, plantain, pomegranate, poplar, radiata pine, radicchio,Southern pine, sweetgum, tangerine, triticale, vine, yams, apple, pear,quince, cherry, apricot, melon, hemp, buckwheat, grape, raspberry,chenopodium, blueberry, nectarine, peach, plum, strawberry, watermelon,eggplant, pepper, cauliflower, Brassica, e.g., broccoli, cabbage,ultilan sprouts, onion, carrot, leek, beet, broad bean, celery, radish,pumpkin, endive, gourd, garlic, snapbean, spinach, squash, turnip,ultilane, and zucchini.

Yet other sources of nucleic acid molecules are ornamental plantsincluding, but not limited to, impatiens, Begonia, Pelargonium, Viola,Cyclamen, Verbena, Vinca, Tagetes, Primula, Saint Paulia, Agertum,Amaranthus, Antihirrhinum, Aquilegia, Cineraria, Clover, Cosmo, Cowpea,Dahlia, Datura, Delphinium, Gerbera, Gladiolus, Gloxinia, Hippeastrum,Mesembryanthemum, Salpiglossos, and Zinnia, and plants such as thoseshown below.

COMMON MAP REFERENCES FAMILY LATIN NAME NAME RESOURCES CucurbitaceaeCucumis sativus Cucumber Cucumis melo Melon Citrullus lanatus WatermelonCucurbita pepo Squash- summer Cucurbita maxima Squash- winter CucurbitaPumpkin/ moschata butternut Solanaceae Lycopersicon Tomato 15x BAC onvariety Heinz 1706 order esculentum from Clemson Genome center 11.6x BACof L. cheesmanii (originates from J. Giovannoni) available from Clemsongenome center EST collection from TIGR EST collection from ClemsomGenome Center TAG 99: 254-271, 1999 (esculentum × pennelli) TAG 89:1007-1013, 1994 (peruvianum) Plant Cell Reports 12: 293-297, 1993(RAPDs) Genetics 132: 1141-1160, 1992 (potato × tomato) Genetics 120:1095-1105, 1988 (RFLP potato and tomato) Genetics 115: 387-393, 1986(esculentum × pennelli isozyme and cDNAs) Capsicum annuum PepperCapsicum Chile pepper frutescens Solanum Eggplant melongena (Nicotiana(Tobacco) tabacum) (Solanum (Potato) tuberosum) (Petunia x hybrida(Petunia) 4x BAC of Petunia hybrida 7984 hort. Ex E. Vilm) availablefrom Clemson genome center Brassicaceae Brassica oleracea Broccoli L.var. italica Brassica oleracea Cabbage L. var. capitata Brassica rapaChinese Cabbage Brassica oleracea Cauliflower L. var. botrytis Raphanussativus Daikon var. niger (Brassica napus) (Oilseed rape) Arabidopsis12x and 6x BACs on Columbia strain available from Clemson genome centerUmbelliferae Daucus carota Carrot Compositae Lactuca sativa LettuceHelianthus annuus (Sunflower) Chenopodiaceae Spinacia oleracea Spinach(Beta vulgaris) (Sugar Beet) Leguminosae Phaseolus vulgaris Bean 4.3xBAC available from Clemson genome center Pisum sativum Pea (Glycine max)(Soybean) 7.5x and 7.9x BACs available from Clemson genome centerGramineae Zea mays Sweet Corn Novartis BACs for Mo17 and B73 have beendonated to Clemson Genome Center (Zea mays) (Field Corn) LiliaceaeAllium cepa Onion Leek (Garlic) (Asparagus)

Yet other preferred sources include, but are not limited to, crop plantsand in particular cereals (for example, corn, alfalfa, sunflower,Brassica, canola, soybean, barley, soybean, sugarbeet, cotton,safflower, peanut, sorghum, wheat, millet, tobacco, and the like), andeven more preferably corn, wheat and soybean.

According to one embodiment, the present invention is directed to anucleic acid molecule comprising a nucleotide sequence isolated orobtained from any plant which encodes a polypeptide having at least 70%amino acid sequence identity to a polypeptide encoded by a genecomprising any one of SEQ ID NOs:1-2672, 5959, 5972, 5973, 5977-5990 and6001. Based on the Oryza nucleic acid sequences of the presentinvention, orthologs may be identified or isolated from the genome ofany desired organism, preferably from another plant, according to wellknown techniques based on their sequence similarity to the Oryza nucleicacid sequences, e.g., hybridization, PCR or computer generated sequencecomparisons. For example, all or a portion of a particular Oryza nucleicacid sequence is used as a probe that selectively hybridizes to othergene sequences present in a population of cloned genomic DNA fragmentsor cDNA fragments (i.e., genomic or cDNA libraries) from a chosen sourceorganism. Further, suitable genomic and cDNA libraries may be preparedfrom any cell or tissue of an organism. Such techniques includehybridization screening of plated DNA libraries (either plaques orcolonies; see, e.g., Sambrook et al., 1989) and amplification by PCRusing oligonucleotide primers preferably corresponding to sequencedomains conserved among related polypeptide or subsequences of thenucleotide sequences provided herein (see, e.g., Innis et al., 1990).These methods are particularly well suited to the isolation of genesequences from organisms closely related to the organism from which theprobe sequence is derived. The application of these methods using theOryza sequences as probes is well suited for the isolation of genesequences from any source organism, preferably other plant species. In aPCR approach, oligonucleotide primers can be designed for use in PCRreactions to amplify corresponding DNA sequences from cDNA or genomicDNA extracted from any plant of interest. Methods for designing PCRprimers and PCR cloning are generally known in the art.

In hybridization techniques, all or part of a known nucleotide sequenceis used as a probe that selectively hybridizes to other correspondingnucleotide sequences present in a population of cloned genomic DNAfragments or cDNA fragments (i.e., genomic or cDNA libraries) from achosen organism. The hybridization probes may be genomic DNA fragments,cDNA fragments, RNA fragments, or other oligonucleotides, and may belabeled with a detectable group such as ³²P, or any other detectablemarker. Thus, for example, probes for hybridization can be made bylabeling synthetic oligonucleotides based on the sequence of theinvention. Methods for preparation of probes for hybridization and forconstruction of cDNA and genomic libraries are generally known in theart and are disclosed in Sambrook et al. (1989). In general, sequencesthat hybridize to the sequences disclosed herein will have at least 40%to 50%, about 60% to 70% and even about 80% 85%, 90%, 95% to 98% or moreidentity with the disclosed sequences. That is, the sequence similarityof sequences may range, sharing at least about 40% to 50%, about 60% to70%, and even about 80%, 85%, 90%, 95% to 98% sequence similarity.

The nucleic acid molecules of the invention can also be identified by,for example, a search of known databases for genes encoding polypeptideshaving a specified amino acid sequence identity or DNA having aspecified nucleotide sequence identity. Methods of alignment ofsequences for comparison are well known in the art and are describedhereinabove.

Virtually any DNA composition may be used for delivery to recipientplant cells, e.g., monocotyledonous cells, to ultimately produce fertiletransgenic plants in accordance with the present invention. For example,DNA segments or fragments in the form of vectors and plasmids, or linearDNA segments or fragments, in some instances containing only the DNAelement to be expressed in the plant, and the like, may be employed. Theconstruction of vectors which may be employed in conjunction with thepresent invention will be known to those of skill of the art in light ofthe present disclosure (see, e.g., Sambrook et al., 1989; Gelvin et al.,1990).

Vectors, plasmids, cosmids, YACs (yeast artificial chromosomes), BACs(bacterial artificial chromosomes) and DNA segments for use intransforming such cells will, of course, generally comprise the cDNA,gene or genes which one desires to introduce into the cells. These DNAconstructs can further include structures such as promoters, enhancers,polylinkers, or even regulatory genes as desired. The DNA segment,fragment or gene chosen for cellular introduction will often encode aprotein which will be expressed in the resultant recombinant cells, suchas will result in a screenable or selectable trait and/or which willimpart an improved phenotype to the regenerated plant. However, this maynot always be the case, and the present invention also encompassestransgenic plants incorporating non-expressed transgenes.

In certain embodiments, it is contemplated that one may wish to employreplication-competent viral vectors in monocot transformation. Suchvectors include, for example, wheat dwarf virus (WDV) “shuttle” vectors,such as pW1-11 and PW1-GUS (Ugaki et al., 1991). These vectors arecapable of autonomous replication in maize cells as well as E. coli, andas such may provide increased sensitivity for detecting DNA delivered totransgenic cells. A replicating vector may also be useful for deliveryof genes flanked by DNA sequences from transposable elements such as Ac,Ds, or Mu. It has been proposed (Laufs et al., 1990) that transpositionof these elements within the maize genome requires DNA replication. Itis also contemplated that transposable elements would be useful forintroducing DNA segments or fragments lacking elements necessary forselection and maintenance of the plasmid vector in bacteria, e.g.,antibiotic resistance genes and origins of DNA replication. It is alsoproposed that use of a transposable element such as Ac, Ds, or Mu wouldactively promote integration of the desired DNA and hence increase thefrequency of stably transformed cells. The use of a transposable elementsuch as Ac, Ds, or Mu may actively promote integration of the DNA ofinterest and hence increase the frequency of stably transformed cells.Transposable elements may be useful to allow separation of genes ofinterest from elements necessary for selection and maintenance of aplasmid vector in bacteria or selection of a transformant. By use of atransposable element, desirable and undesirable DNA sequences may betransposed apart from each other in the genome, such that throughgenetic segregation in progeny, one may identify plants with either thedesirable undesirable DNA sequences.

It is one of the objects of the present invention to provide recombinantDNA molecules comprising a nucleotide sequence which directstranscription according to the invention operably linked to a nucleicacid segment or sequence of interest. The nucleic acid segment ofinterest can, for example, code for a ribosomal RNA, an antisense RNA orany other type of RNA that is not translated into protein. In anotherpreferred embodiment of the invention, the nucleic acid segment ofinterest is translated into a protein product. The nucleotide sequencewhich directs transcription and/or the nucleic acid segment may be ofhomologous or heterologous origin with respect to the plant to betransformed. A recombinant DNA molecule useful for introduction intoplant cells includes that which has been derived or isolated from anysource, that may be subsequently characterized as to structure, sizeand/or function, chemically altered, and later introduced into plants.An example of a nucleotide sequence or segment of interest “derived”from a source, would be a nucleotide sequence or segment that isidentified as a useful fragment within a given organism, and which isthen chemically synthesized in essentially pure form. An example of sucha nucleotide sequence or segment of interest “isolated” from a source,would be nucleotide sequence or segment that is excised or removed fromsaid source by chemical means, e.g., by the use of restrictionendonucleases, so that it can be further manipulated, e.g., amplified,for use in the invention, by the methodology of genetic engineering.Such a nucleotide sequence or segment is commonly referred to as“recombinant.”

Therefore a useful nucleotide sequence, segment or fragment of interestincludes completely synthetic DNA, semi-synthetic DNA, DNA isolated frombiological sources, and DNA derived from introduced RNA. Generally, theintroduced DNA is not originally resident in the plant genotype which isthe recipient of the DNA, but it is within the scope of the invention toisolate a gene from a given plant genotype, and to subsequentlyintroduce multiple copies of the gene into the same genotype, e.g., toenhance production of a given gene product such as a storage protein ora protein that confers tolerance or resistance to water deficit.

The introduced recombinant DNA molecule includes but is not limited to,DNA from plant genes, and non-plant genes such as those from bacteria,yeasts, animals or viruses. The introduced DNA can include modifiedgenes, portions of genes, or chimeric genes, including genes from thesame or different genotype. The term “chimeric gene” or “chimeric DNA”is defined as a gene or DNA sequence or segment comprising at least twoDNA sequences or segments from species which do not combine DNA undernatural conditions, or which DNA sequences or segments are positioned orlinked in a manner which does not normally occur in the native genome ofuntransformed plant.

The introduced recombinant DNA molecule used for transformation hereinmay be circular or linear, double-stranded or single-stranded.Generally, the DNA is in the form of chimeric DNA, such as plasmid DNA,that can also contain coding regions flanked by regulatory sequenceswhich promote the expression of the recombinant DNA present in theresultant plant.

Generally, the introduced recombinant DNA molecule will be relativelysmall, i.e., less than about 30 kb to minimize any susceptibility tophysical, chemical, or enzymatic degradation which is known to increaseas the size of the nucleotide molecule increases. As noted above, thenumber of proteins, RNA transcripts or mixtures thereof which isintroduced into the plant genome is preferably preselected and defined,e.g., from one to about 5-10 such products of the introduced DNA may beformed.

Two principal methods for the control of expression are known, viz.:overexpression and underexpression. Overexpression can be achieved byinsertion of one or more than one extra copy of the selected gene. Itis, however, not unknown for plants or their progeny, originallytransformed with one or more than one extra copy of a nucleotidesequence, to exhibit the effects of underexpression as well asoverexpression. For underexpression there are two principle methodswhich are commonly referred to in the art as “antisense downregulation”and “sense downregulation” (sense downregulation is also referred to as“cosuppression”). Generically these processes are referred to as “genesilencing”. Both of these methods lead to an inhibition of expression ofthe target gene.

Obtaining sufficient levels of transgene expression in the appropriateplant tissues is an important aspect in the production of geneticallyengineered crops. Expression of heterologous DNA sequences in a planthost is dependent upon the presence of an operably linked promoter thatis functional within the plant host. Choice of the promoter sequencewill determine when and where within the organism the heterologous DNAsequence is expressed.

It is specifically contemplated by the inventors that one couldmutagenize a promoter to potentially improve the utility of the elementsfor the expression of transgenes in plants. The mutagenesis of theseelements can be carried out at random and the mutagenized promotersequences screened for activity in a trial-by-error procedure.

Alternatively, particular sequences which provide the promoter withdesirable expression characteristics, or the promoter with expressionenhancement activity, could be identified and these or similar sequencesintroduced into the sequences via mutation. It is further contemplatedthat one could mutagenize these sequences in order to enhance theirexpression of transgenes in a particular species.

The means for mutagenizing a DNA segment encoding a promoter sequence ofthe current invention are well-known to those of skill in the art. Asindicated, modifications to promoter or other regulatory element may bemade by random, or site-specific mutagenesis procedures. The promoterand other regulatory element may be modified by altering their structurethrough the addition or deletion of one or more nucleotides from thesequence which encodes the corresponding un-modified sequences.

Mutagenesis may be performed in accordance with any of the techniquesknown in the art, such as, and not limited to, synthesizing anoligonucleotide having one or more mutations within the sequence of aparticular regulatory region. In particular, site-specific mutagenesisis a technique useful in the preparation of promoter mutants, throughspecific mutagenesis of the underlying DNA. The technique furtherprovides a ready ability to prepare and test sequence variants, forexample, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed.

Typically, a primer of about 17 to about 75 nucleotides or more inlength is preferred, with about 10 to about 25 or more residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as exemplified by various publications. As will be appreciated,the technique typically employs a phage vector which exists in both asingle stranded and double stranded form. Typical vectors useful insite-directed mutagenesis include vectors such as the M13 phage. Thesephage are readily commercially available and their use is generally wellknown to those skilled in the art.

Double stranded plasmids also are routinely employed in site directedmutagenesis which eliminates the step of transferring the gene ofinterest from a plasmid to a phage.

In general, site-directed mutagenesis in accordance herewith isperformed by first obtaining a single-stranded vector or melting apartof two strands of a double stranded vector which includes within itssequence a DNA sequence which encodes the promoter. An oligonucleotideprimer bearing the desired mutated sequence is prepared, generallysynthetically. This primer is then annealed with the single-strandedvector, and subjected to DNA polymerizing enzymes such as E. colipolymerase I Klenow fragment, in order to complete the synthesis of themutation-bearing strand. Thus, a heteroduplex is formed wherein onestrand encodes the original non-mutated sequence and the second strandbears the desired mutation.

This heteroduplex vector is then used to transform or transfectappropriate cells, such as E. coli cells, and cells are selected whichinclude recombinant vectors bearing the mutated sequence arrangement.Vector DNA can then be isolated from these cells and used for planttransformation. A genetic selection scheme was devised by Kunkel et al.(1987) to enrich for clones incorporating mutagenic oligonucleotides.Alternatively, the use of PCR with commercially available thermostableenzymes such as Taq polymerase may be used to incorporate a mutagenicoligonucleotide primer into an amplified DNA fragment that can then becloned into an appropriate cloning or expression vector. ThePCR-mediated mutagenesis procedures of Tomic et al. (1990) and Upenderet al. (1995) provide two examples of such protocols. A PCR employing athermostable ligase in addition to a thermostable polymerase also may beused to incorporate a phosphorylated mutagenic oligonucleotide into anamplified DNA fragment that may then be cloned into an appropriatecloning or expression vector. The mutagenesis procedure described byMichael (1994) provides an example of one such protocol.

The preparation of sequence variants of the selected promoter-encodingDNA segments using site-directed mutagenesis is provided as a means ofproducing potentially useful species and is not meant to be limiting asthere are other ways in which sequence variants of DNA sequences may beobtained. For example, recombinant vectors encoding the desired promotersequence may be treated with mutagenic agents, such as hydroxylamine, toobtain sequence variants.

As used herein, the term “oligonucleotide directed mutagenesisprocedure” refers to template-dependent processes and vector-mediatedpropagation which result in an increase in the concentration of aspecific nucleic acid molecule relative to its initial concentration, orin an increase in the concentration of a detectable signal, such asamplification. As used herein, the term “oligonucleotide directedmutagenesis procedure” also is intended to refer to a process thatinvolves the template-dependent extension of a primer molecule. The termtemplate-dependent process refers to nucleic acid synthesis of an RNA ora DNA molecule wherein the sequence of the newly synthesized strand ofnucleic acid is dictated by the well-known rules of complementary basepairing (see, for example, Watson and Rarnstad, 1987). Typically, vectormediated methodologies involve the introduction of the nucleic acidfragment into a DNA or RNA vector, the clonal amplification of thevector, and the recovery of the amplified nucleic acid fragment.Examples of such methodologies are provided by U.S. Pat. No. 4,237,224.A number of template dependent processes are available to amplify thetarget sequences of interest present in a sample, such methods beingwell known in the art and specifically disclosed herein below.

Where a clone comprising a promoter has been isolated in accordance withthe instant invention, one may wish to delimit the essential promoterregions within the clone. One efficient, targeted means for preparingmutagenizing promoters relies upon the identification of putativeregulatory elements within the promoter sequence. This can be initiatedby comparison with promoter sequences known to be expressed in similartissue-specific or developmentally unique manner. Sequences which areshared among promoters with similar expression patterns are likelycandidates for the binding of transcription factors and are thus likelyelements which confer expression patterns. Confirmation of theseputative regulatory elements can be achieved by deletion analysis ofeach putative regulatory region followed by functional analysis of eachdeletion construct by assay of a reporter gene which is functionallyattached to each construct. As such, once a starting promoter sequenceis provided, any of a number of different deletion mutants of thestarting promoter could be readily prepared.

As indicated above, deletion mutants, deletion mutants of the promoterof the invention also could be randomly prepared and then assayed. Withthis strategy, a series of constructs are prepared, each containing adifferent portion of the clone (a subclone), and these constructs arethen screened for activity. A suitable means for screening for activityis to attach a deleted promoter or intron construct which contains adeleted segment to a selectable or screenable marker, and to isolateonly those cells expressing the marker gene. In this way, a number ofdifferent, deleted promoter constructs are identified which still retainthe desired, or even enhanced, activity. The smallest segment which isrequired for activity is thereby identified through comparison of theselected constructs. This segment may then be used for the constructionof vectors for the expression of exogenous genes.

Furthermore, it is contemplated that promoters combining elements frommore than one promoter may be useful. For example, U.S. Pat. No.5,491,288 discloses combining a Cauliflower Mosaic Virus promoter with ahistone promoter. Thus, the elements from the promoters disclosed hereinmay be combined with elements from other promoters.

Promoters which are useful for plant transgene expression include thosethat are inducible, viral, synthetic, constitutive (Odell et al., 1985),temporally regulated, spatially regulated, tissue-specific, andspatio-temporally regulated.

Where expression in specific tissues or organs is desired,tissue-specific promoters may be used. In contrast, where geneexpression in response to a stimulus is desired, inducible promoters arethe regulatory elements of choice. Where continuous expression isdesired throughout the cells of a plant, constitutive promoters areutilized. Additional regulatory sequences upstream and/or downstreamfrom the core promoter sequence may be included in expression constructsof transformation vectors to bring about varying levels of expression ofheterologous nucleotide sequences in a transgenic plant.

A variety of 5N and 3N transcriptional regulatory sequences areavailable for use in the present invention. Transcriptional terminatorsare responsible for the termination of transcription and correct mRNApolyadenylation. The 3N nontranslated regulatory DNA sequence preferablyincludes from about 50 to about 1,000, more preferably about 100 toabout 1,000, nucleotide base pairs and contains plant transcriptionaland translational termination sequences. Appropriate transcriptionalterminators and those which are known to function in plants include theCaMV 35S terminator, the tml terminator, the nopaline synthaseterminator, the pea rbcS E9 terminator, the terminator for the T7transcript from the octopine synthase gene of Agrobacterium tumefaciens,and the 3N end of the protease inhibitor I or II genes from potato ortomato, although other 3N elements known to those of skill in the artcan also be employed. Alternatively, one also could use a gamma coixin,oleosin 3 or other terminator from the genus Coix. Preferred 3N elementsinclude those from the nopaline synthase gene of Agrobacteriumtumefaciens (Bevan et al., 1983), the terminator for the T7 transcriptfrom the octopine synthase gene of Agrobacterium tumefaciens, and the 3′end of the protease inhibitor I or II genes from potato or tomato.

As the DNA sequence between the transcription initiation site and thestart of the coding sequence, i.e., the untranslated leader sequence,can influence gene expression, one may also wish to employ a particularleader sequence. Preferred leader sequences are contemplated to includethose which include sequences predicted to direct optimum expression ofthe attached gene, i.e., to include a preferred consensus leadersequence which may increase or maintain mRNA stability and preventinappropriate initiation of translation. The choice of such sequenceswill be known to those of skill in the art in light of the presentdisclosure. Sequences that are derived from genes that are highlyexpressed in plants will be most preferred.

Other sequences that have been found to enhance gene expression intransgenic plants include intron sequences (e.g., from Adh1, bronze1,actin1, actin 2 (WO 00/760067), or the sucrose synthase intron) andviral leader sequences (e.g., from TMV, MCMV and AMV). For example, anumber of non-translated leader sequences derived from viruses are knownto enhance expression. Specifically, leader sequences from TobaccoMosaic Virus (TMV), Maize Chlorotic Mottle Virus (MCMV), and AlfalfaMosaic Virus (AMV) have been shown to be effective in enhancingexpression (e.g., Gallie et al., 1987; Skuzeski et al., 1990). Otherleaders known in the art include but are not limited to: Picornavirusleaders, for example, EMCV leader (Encephalomyocarditis 5 noncodingregion) (Elroy-Stein et al., 1989); Potyvirus leaders, for example, TEVleader (Tobacco Etch Virus); MDMV leader (Maize Dwarf Mosaic Virus);Human immunoglobulin heavy-chain binding protein (BiP) leader, (Macejaket al., 1991); Untranslated leader from the coat protein mRNA of alfalfamosaic virus (AMV RNA 4), (Jobling et al., 1987; Tobacco mosaic virusleader (TMV), (Gallie et al., 1989; and Maize Chlorotic Mottle Virusleader (MCMV) (Lommel et al., 1991. See also, Della-Cioppa et al., 1987.

Regulatory elements such as Adh intron 1 (Callis et al., 1987), sucrosesynthase intron (Vasil et al., 1989) or TMV omega element (Gallie, etal., 1989), may further be included where desired.

Examples of enhancers include elements from the CaMV 35S promoter,octopine synthase genes (Ellis el al., 1987), the rice actin I gene, themaize alcohol dehydrogenase gene (Callis et al., 1987), the maizeshrunken I gene (Vasil et al., 1989), TMV Omega element (Gallie et al.,1989) and promoters from non-plant eukaryotes (e.g. yeast; Ma et al.,1988).

Vectors for use in accordance with the present invention may beconstructed to include the ocs enhancer element. This element was firstidentified as a 16 bp palindromic enhancer from the octopine synthase(ocs) gene of ultilane (Ellis et al., 1987), and is present in at least10 other promoters (Bouchez et al., 1989). The use of an enhancerelement, such as the ocs element and particularly multiple copies of theelement, will act to increase the level of transcription from adjacentpromoters when applied in the context of monocot transformation.

Ultimately, the most desirable DNA segments for introduction into, forexample, a monocot genome, may be homologous genes or gene familieswhich encode a desired trait (e.g., increased yield per acre) and whichare introduced under the control of novel promoters or enhancers, etc.,or perhaps even homologous or tissue specific (e.g., root-,collar/sheath-, whorl-, stalk-, earshank-, kernel- or leaf-specific)promoters or control elements. Indeed, it is envisioned that aparticular use of the present invention will be the expression of a genein a constitutive or a seed-specific manner.

Vectors for use in tissue-specific targeting of genes in transgenicplants will typically include tissue-specific promoters and may alsoinclude other tissue-specific control elements such as enhancersequences. Promoters which direct specific or enhanced expression incertain plant tissues will be known to those of skill in the art inlight of the present disclosure. These include, for example, the rbcSpromoter, specific for green tissue; the ocs, nos and mas promoterswhich have higher activity in roots or wounded leaf tissue; a truncated(−90 to +8) 35S promoter which directs enhanced expression in roots, analpha-tubulin gene that directs expression in roots and promotersderived from zein storage protein genes which direct expression inendosperm. It is particularly contemplated that one may advantageouslyuse the 16 bp ocs enhancer element from the octopine synthase (ocs) gene(Ellis et al., 1987; Bouchez et al., 1989), especially when present inmultiple copies, to achieve enhanced expression in roots.

Tissue specific expression may be functionally accomplished byintroducing a constitutively expressed gene (all tissues) in combinationwith an antisense gene that is expressed only in those tissues where thegene product is not desired. For example, a gene coding for the crystaltoxin protein from B. thuringiensis (Bt) may be introduced such that itis expressed in all tissues using the 35S promoter from CauliflowerMosaic Virus. Expression of an antisense transcript of the Bt gene in amaize kernel, using for example a zein promoter, would preventaccumulation of the Bt protein in seed. Hence the protein encoded by theintroduced gene would be present in all tissues except the kernel.

Expression of some genes in transgenic plants will be desired only underspecified conditions. For example, it is proposed that expression ofcertain genes that confer resistance to environmental stress factorssuch as drought will be desired only under actual stress conditions. Itis contemplated that expression of such genes throughout a plantsdevelopment may have detrimental effects. It is known that a largenumber of genes exist that respond to the environment. For example,expression of some genes such as rbcS, encoding the small subunit ofribulose bisphosphate carboxylase, is regulated by light as mediatedthrough phytochrome. Other genes are induced by secondary stimuli. Forexample, synthesis of abscisic acid (ABA) is induced by certainenvironmental factors, including but not limited to water stress. Anumber of genes have been shown to be induced by ABA (Skriver and Mundy,1990). It is also anticipated that expression of genes conferringresistance to insect predation would be desired only under conditions ofactual insect infestation. Therefore, for some desired traits inducibleexpression of genes in transgenic plants will be desired.

Expression of a gene in a transgenic plant will be desired only in acertain time period during the development of the plant. Developmentaltiming is frequently correlated with tissue specific gene expression.For example, expression of zein storage proteins is initiated in theendosperm about 15 days after pollination.

Additionally, vectors may be constructed and employed in theintracellular targeting of a specific gene product within the cells of atransgenic plant or in directing a protein to the extracellularenvironment. This will generally be achieved by joining a DNA sequenceencoding a transit or signal peptide sequence to the coding sequence ofa particular gene. The resultant transit, or signal, peptide willtransport the protein to a particular intracellular, or extracellulardestination, respectively, and will then be post-translationallyremoved. Transit or signal peptides act by facilitating the transport ofproteins through intracellular membranes, e.g., vacuole, vesicle,plastid and mitochondrial membranes, whereas signal peptides directproteins through the extracellular membrane.

A particular example of such a use concerns the direction of a herbicideresistance gene, such as the EPSPS gene, to a particular organelle suchas the chloroplast rather than to the cytoplasm. This is exemplified bythe use of the rbcs transit peptide which confers plastid-specifictargeting of proteins. In addition, it is proposed that it may bedesirable to target certain genes responsible for male sterility to themitochondria, or to target certain genes for resistance tophytopathogenic organisms to the extracellular spaces, or to targetproteins to the vacuole.

By facilitating the transport of the protein into compartments insideand outside the cell, these sequences may increase the accumulation ofgene product protecting them from proteolytic degradation. Thesesequences also allow for additional mRNA sequences from highly expressedgenes to be attached to the coding sequence of the genes. Since mRNAbeing translated by ribosomes is more stable than naked mRNA, thepresence of translatable mRNA in front of the gene may increase theoverall stability of the mRNA transcript from the gene and therebyincrease synthesis of the gene product. Since transit and signalsequences are usually post-translationally removed from the initialtranslation product, the use of these sequences allows for the additionof extra translated sequences that may not appear on the finalpolypeptide. Targeting of certain proteins may be desirable in order toenhance the stability of the protein (U.S. Pat. No. 5,545,818).

It may be useful to target DNA itself within a cell. For example, it maybe useful to target introduced DNA to the nucleus as this may increasethe frequency of transformation. Within the nucleus itself it would beuseful to target a gene in order to achieve site specific integration.For example, it would be useful to have an gene introduced throughtransformation replace an existing gene in the cell.

Other elements include those that can be regulated by endogenous orexogenous agents, e.g., by zinc finger proteins, including naturallyoccurring zinc finger proteins or chimeric zinc finger proteins (see,e.g., U.S. Pat. No. 5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO98/53057; WO 98/53058; WO 00/23464; WO 95/19431; and WO 98/54311) ormyb-like transcription factors. For example, a chimeric zinc fingerprotein may include amino acid sequences which bind to a specific DNAsequence (the zinc finger) and amino acid sequences that activate (e.g.,GAL 4 sequences) or repress the transcription of the sequences linked tothe specific DNA sequence.

It is one of the objects of the present invention to provide recombinantDNA molecules comprising a nucleotide sequence according to theinvention operably linked to a nucleotide segment of interest.

A nucleotide segment of interest is reflective of the commercial marketsand interests of those involved in the development of the crop. Cropsand markets of interest changes, and as developing nations open up worldmarkets, new crops and technologies will also emerge. In addition, asthe understanding of agronomic traits and characteristics such as yieldand heterosis increase, the choice of genes for transformation willchange accordingly. General categories of nucleotides of interestinclude, for example, genes involved in information, such as zincfingers, those involved in communication, such as kinases, and thoseinvolved in housekeeping, such as heat shock proteins. More specificcategories of transgenes, for example, include genes encoding importanttraits for agronomics, insect resistance, disease resistance, herbicideresistance, sterility, grain characteristics, and commercial products.Genes of interest include, generally, those involved in starch, oil,carbohydrate, or nutrient metabolism, as well as those affecting kernelsize, sucrose loading, zinc finger proteins, see, e.g., U.S. Pat. No.5,789,538, WO 99/48909; WO 99/45132; WO 98/53060; WO 98/53057; WO98/53058; WO 00/23464; WO 95/19431; and WO 98/54311, and the like.

One skilled in the art recognizes that the expression level andregulation of a transgene in a plant can vary significantly from line toline. Thus, one has to test several lines to find one with the desiredexpression level and regulation. Once a line is identified with thedesired regulation specificity of a chimeric Cre transgene, it can becrossed with lines carrying different inactive replicons or inactivetransgene for activation.

Other sequences which may be linked to the gene of interest whichencodes a polypeptide are those which can target to a specificorganelle, e.g., to the mitochondria, nucleus, or plastid, within theplant cell. Targeting can be achieved by providing the polypeptide withan appropriate targeting peptide sequence, such as a secretory signalpeptide (for secretion or cell wall or membrane targeting, a plastidtransit peptide, a chloroplast transit peptide, e.g., the chlorophylla/b binding protein, a mitochondrial target peptide, a vacuole targetingpeptide, or a nuclear targeting peptide, and the like. For example, thesmall subunit of ribulose bisphosphate carboxylase transit peptide, theEPSPS transit peptide or the dihydrodipicolinic acid synthase transitpeptide may be used. For examples of plastid organelle targetingsequences (see WO 00/12732). Plastids are a class of plant organellesderived from proplastids and include chloroplasts, leucoplasts,aravloplasts, and chromoplasts. The plastids are major sites ofbiosynthesis in plants. In addition to photosynthesis in thechloroplast, plastids are also sites of lipid biosynthesis, nitratereduction to ammonium, and starch storage. And while plastids containtheir own circular genome, most of the proteins localized to theplastids are encoded by the nuclear genome and are imported into theorganelle from the cytoplasm.

Transgenes used with the present invention will often be genes thatdirect the expression of a particular protein or polypeptide product,but they may also be non-expressible DNA segments, e.g., transposonssuch as Ds that do no direct their own transposition. As used herein, an“expressible gene” is any gene that is capable of being transcribed intoRNA (e.g., mRNA, antisense RNA, etc.) or translated into a protein,expressed as a trait of interest, or the like, etc., and is not limitedto selectable, screenable or non-selectable marker genes. The inventionalso contemplates that, where both an expressible gene that is notnecessarily a marker gene is employed in combination with a marker gene,one may employ the separate genes on either the same or different DNAsegments for transformation. In the latter case, the different vectorsare delivered concurrently to recipient cells to maximizecotransformation.

The choice of the particular DNA segments to be delivered to therecipient cells will often depend on the purpose of the transformation.One of the major purposes of transformation of crop plants is to addsome commercially desirable, agronomically important traits to theplant. Such traits include, but are not limited to, herbicide resistanceor tolerance; insect resistance or tolerance; disease resistance ortolerance (viral, bacterial, fungal, nematode); stress tolerance and/orresistance, as exemplified by resistance or tolerance to drought, heat,chilling, freezing, excessive moisture, salt stress; oxidative stress;increased yields; food content and makeup; physical appearance; malesterility; drydown; standability; prolificacy; starch properties; oilquantity and quality; and the like. One may desire to incorporate one ormore genes conferring any such desirable trait or traits, such as, forexample, a gene or genes encoding pathogen resistance.

In certain embodiments, the present invention contemplates thetransformation of a recipient cell with more than one advantageoustransgene. Two or more transgenes can be supplied in a singletransformation event using either distinct transgene-encoding vectors,or using a single vector incorporating two or more gene codingsequences. For example, plasmids bearing the bar and aroA expressionunits in either convergent, divergent, or colinear orientation, areconsidered to be particularly useful. Further preferred combinations arethose of an insect resistance gene, such as a Bt gene, along with aprotease inhibitor gene such as pinII, or the use of bar in combinationwith either of the above genes. Of course, any two or more transgenes ofany description, such as those conferring herbicide, insect, disease(viral, bacterial, fungal, nematode) or drought resistance, malesterility, drydown, standability, prolificacy, starch properties, oilquantity and quality, or those increasing yield or nutritional qualitymay be employed as desired.

A. Exemplary Transgenes

1. Herbicide Resistance

The genes encoding phosphinothricin acetyltransferase (bar and pat),glyphosate tolerant EPSP synthase genes, the glyphosate degradativeenzyme gene gox encoding glyphosate oxidoreductase, deh (encoding adehalogenase enzyme that inactivates dalapon), herbicide resistant(e.g., sulfonylurea and imidazolinone) acetolactate synthase, and bxngenes (encoding a nitrilase enzyme that degrades bromoxynil) are goodexamples of herbicide resistant genes for use in transformation. The barand pat genes code for an enzyme, phosphinothricin acetyltransferase(PAT), which inactivates the herbicide phosphinothricin and preventsthis compound from inhibiting glutamine synthetase enzymes. The enzyme5-enolpyruvylshikimate 3-phosphate synthase (EPSP Synthase), is normallyinhibited by the herbicide N-(phosphonomethyl)glycine (glyphosate).However, genes are known that encode glyphosate-resistant EPSP Synthaseenzymes.

These genes are particularly contemplated for use in monocottransformation. The deh gene encodes the enzyme dalapon dehalogenase andconfers resistance to the herbicide dalapon. The bxn gene codes for aspecific nitrilase enzyme that converts bromoxynil to a non-herbicidaldegradation product.

2. Insect Resistance

An important aspect of the present invention concerns the introductionof insect resistance-conferring genes into plants. Potential insectresistance genes which can be is introduced include Bacillusthuringiensis crystal toxin genes or Bt genes (Watrud et al., 1985). Btgenes may provide resistance to lepidopteran or coleopteran pests suchas European Corn Borer (ECB) and corn rootworm (CRW). Preferred Bt toxingenes for use in such embodiments include the CryIA(b) and CryIA(c)genes. Endotoxin genes from other species of B. thuringiensis whichaffect insect growth or development may also be employed in this regard.

The poor expression of Bt toxin genes in plants is a well-documentedphenomenon, and the use of different promoters, fusion proteins, andleader sequences has not led to significant increases in Bt proteinexpression (Vaeck et al., 1989; Barton et al., 1987). It is thereforecontemplated that the most advantageous Bt genes for use in thetransformation protocols disclosed herein will be those in which thecoding sequence has been modified to effect increased expression inplants, and more particularly, those in which maize preferred codonshave been used. Examples of such modified Bt toxin genes include thevariant Bt CryIA(b) gene termed Iab6 (Perlak et al., 1991) and thesynthetic CryIA(c) genes termed 1800a and 1800b.

Protease inhibitors may also provide insect resistance (Johnson et al.,1989), and will thus have utility in plant transformation. The use of aprotease inhibitor II gene, pinII, from tomato or potato is envisionedto be particularly useful. Even more advantageous is the use of a pinIIgene in combination with a Bt toxin gene, the combined effect of whichhas been discovered by the present inventors to produce synergisticinsecticidal activity. Other genes which encode inhibitors of theinsects' digestive system, or those that encode enzymes or co-factorsthat facilitate the production of inhibitors, may also be useful. Thisgroup may be exemplified by oryzacystatin and amylase inhibitors, suchas those from wheat and barley.

Also, genes encoding lectins may confer additional or alternativeinsecticide properties. Lectins (originally termed phytohemagglutinins)are multivalent carbohydrate-binding proteins which have the ability toagglutinate red blood cells from a range of species. Lectins have beenidentified recently as insecticidal agents with activity againstweevils, ECB and rootworm (Murdock et al., 1990; Czapla and Lang, 1990).Lectin genes contemplated to be useful include, for example, barley andwheat germ agglutinin (WGA) and rice lectins (Gatehouse et al., 1984),with WGA being preferred.

Genes controlling the production of large or small polypeptides activeagainst insects when introduced into the insect pests, such as, e.g.,lytic peptides, peptide hormones and toxins and venoms, form anotheraspect of the invention. For example, it is contemplated that theexpression of juvenile hormone esterase, directed towards specificinsect pests, may also result in insecticidal activity, or perhaps causecessation of metamorphosis (Hammock et al., 1990).

Transgenic plants expressing genes which encode enzymes that affect theintegrity of the insect cuticle form yet another aspect of theinvention. Such genes include those encoding, e.g., chitinase,proteases, lipases and also genes for the production of nikkomycin, acompound that inhibits chitin synthesis, the introduction of any ofwhich is contemplated to produce insect resistant maize plants. Genesthat code for activities that affect insect molting, such thoseaffecting the production of ecdysteroid UDP-glucosyl transferase, alsofall within the scope of the useful transgenes of the present invention.

Genes that code for enzymes that facilitate the production of compoundsthat reduce the nutritional quality of the host plant to insect pestsare also encompassed by the present invention. It may be possible, forinstance, to confer insecticidal activity on a plant by altering itssterol composition. Sterols are obtained by insects from their diet andare used for hormone synthesis and membrane stability. Thereforealterations in plant sterol composition by expression of novel genes,e.g., those that directly promote the production of undesirable sterolsor those that convert desirable sterols into undesirable forms, couldhave a negative effect on insect growth and/or development and henceendow the plant with insecticidal activity. Lipoxygenases are naturallyoccurring plant enzymes that have been shown to exhibit anti-nutritionaleffects on insects and to reduce the nutritional quality of their diet.Therefore, further embodiments of the invention concern transgenicplants with enhanced lipoxygenase activity which may be resistant toinsect feeding.

The present invention also provides methods and compositions by which toachieve qualitative or quantitative changes in plant secondarymetabolites. One example concerns transforming plants to produce DIMBOAwhich, it is contemplated, will confer resistance to European cornborer, rootworm and several other maize insect pests. Candidate genesthat are particularly considered for use in this regard include thosegenes at the bx locus known to be involved in the synthetic DIMBOApathway (Dunn et al., 1981). The introduction of genes that can regulatethe production of maysin, and genes involved in the production ofdhurrin in sorghum, is also contemplated to be of use in facilitatingresistance to earworm and rootworm, respectively.

Tripsacum dactyloides is a species of grass that is resistant to certaininsects, including corn root worm. It is anticipated that genes encodingproteins that are toxic to insects or are involved in the biosynthesisof compounds toxic to insects will be isolated from Tripsacum and thatthese novel genes will be useful in conferring resistance to insects. Itis known that the basis of insect resistance in Tripsacum is genetic,because said resistance has been transferred to Zea mays via sexualcrosses (Branson and Guss, 1972).

Further genes encoding proteins characterized as having potentialinsecticidal activity may also be used as transgenes in accordanceherewith. Such genes include, for example, the cowpea trypsin inhibitor(CpTI; Hilder et al., 1987) which may be used as a rootworm deterrent;genes encoding avermectin (Campbell, 1989; Ikeda et al., 1987) which mayprove particularly useful as a corn rootworm deterrent; ribosomeinactivating protein genes; and even genes that regulate plantstructures. Transgenic maize including anti-insect antibody genes andgenes that code for enzymes that can covert a non-toxic insecticide(pro-insecticide) applied to the outside of the plant into aninsecticide inside the plant are also contemplated.

3. Environment or Stress Resistance

Improvement of a plant's ability to tolerate various environmentalstresses such as, but not limited to, drought, excess moisture,chilling, freezing, high temperature, salt, and oxidative stress, canalso be effected through expression of heterologous, or overexpressionof homologous genes. Benefits may be realized in terms of increasedresistance to freezing temperatures through the introduction of an“antifreeze” protein such as that of the Winter Flounder (Cutler et al.,1989) or synthetic gene derivatives thereof. Improved chilling tolerancemay also be conferred through increased expression ofglycerol-3-phosphate acetyltransferase in chloroplasts (Murata et al.,1992; Wolter et al., 1992). Resistance to oxidative stress (oftenexacerbated by conditions such as chilling temperatures in combinationwith high light intensities) can be conferred by expression ofsuperoxide dismutase (Gupta et al., 1993), and may be improved byglutathione reductase (Bowler et al., 1992). Such strategies may allowfor tolerance to freezing in newly emerged fields as well as extendinglater maturity higher yielding varieties to earlier relative maturityzones.

Expression of novel genes that favorably effect plant water content,total water potential, osmotic potential, and turgor can enhance theability of the plant to tolerate drought. As used herein, the terms“drought resistance” and “drought tolerance” are used to refer to aplants increased resistance or tolerance to stress induced by areduction in water availability, as compared to normal circumstances,and the ability of the plant to function and survive in lower-waterenvironments, and perform in a relatively superior manner. In thisaspect of the invention it is proposed, for example, that the expressionof a gene encoding the biosynthesis of osmotically-active solutes canimpart protection against drought. Within this class of genes are DNAsencoding mannitol dehydrogenase (Lee and Saier, 1982) andtrehalose-6-phosphate synthase (Kaasen et al., 1992). Through thesubsequent action of native phosphatases in the cell or by theintroduction and coexpression of a specific phosphatase, theseintroduced genes will result in the accumulation of either mannitol ortrehalose, respectively, both of which have been well documented asprotective compounds able to mitigate the effects of stress. Mannitolaccumulation in transgenic tobacco has been verified and preliminaryresults indicate that plants expressing high levels of this metaboliteare able to tolerate an applied osmotic stress (Tarczynski et al.,1992).

Similarly, the efficacy of other metabolites in protecting either enzymefunction (e.g. alanopine or propionic acid) or membrane integrity (e.g.,alanopine) has been documented (Loomis et al., 1989), and thereforeexpression of gene encoding the biosynthesis of these compounds canconfer drought resistance in a manner similar to or complimentary tomannitol. Other examples of naturally occurring metabolites that areosmotically active and/or provide some direct protective effect duringdrought and/or desiccation include sugars and sugar derivatives such asfructose, erythritol (Coxson et al., 1992), sorbitol, dulcitol (Karstenet al., 1992), glucosylglycerol (Reed et al., 1984; Erdmann et al.,1992), sucrose, stachyose (Koster and Leopold, 1988; Blackman et al.,1992), ononitol and pinitol (Vernon and Bohnert, 1992), and raffinose(Bernal-Lugo and Leopold, 1992). Other osmotically active solutes whichare not sugars include, but are not limited to, proline andglycine-betaine (Wyn-Jones and Storey, 1981). Continued canopy growthand increased reproductive fitness during times of stress can beaugmented by introduction and expression of genes such as thosecontrolling the osmotically active compounds discussed above and othersuch compounds, as represented in one exemplary embodiment by the enzymemyoinositol 0-methyltransferase.

It is contemplated that the expression of specific proteins may alsoincrease drought tolerance. Three classes of Late Embryogenic Proteinshave been assigned based on structural similarities (see Dure et al.,1989). All three classes of these proteins have been demonstrated inmaturing (i.e., desiccating) seeds. Within these 3 types of proteins,the Type-II (dehydrin-type) have generally been implicated in droughtand/or desiccation tolerance in vegetative plant parts (i.e. Mundy andChua, 1988; Piatkowski et al., 1990; Yamaguchi-Shinozaki et al., 1992).Recently, expression of a Type-III LEA (HVA-1) in tobacco was found toinfluence plant height, maturity and drought tolerance (Fitzpatrick,1993). Expression of structural genes from all three groups maytherefore confer drought tolerance. Other types of proteins inducedduring water stress include thiol proteases, aldolases and transmembranetransporters (Guerrero et al., 1990), which may confer variousprotective and/or repair-type functions during drought stress. Theexpression of a gene that effects lipid biosynthesis and hence membranecomposition can also be useful in conferring drought resistance on theplant.

Many genes that improve drought resistance have complementary modes ofaction. Thus, combinations of these genes might have additive and/orsynergistic effects in improving drought resistance in maize. Many ofthese genes also improve freezing tolerance (or resistance); thephysical stresses incurred during freezing and drought are similar innature and may be mitigated in similar fashion. Benefit may be conferredvia constitutive expression of these genes, but the preferred means ofexpressing these novel genes may be through the use of a turgor-inducedpromoter (such as the promoters for the turgor-induced genes describedin Guerrero et al. 1990 and Shagan et al., 1993). Spatial and temporalexpression patterns of these genes may enable maize to better withstandstress.

Expression of genes that are involved with specific morphological traitsthat allow for increased water extractions from drying soil would be ofbenefit. For example, introduction and expression of genes that alterroot characteristics may enhance water uptake. Expression of genes thatenhance reproductive fitness during times of stress would be ofsignificant value. For example, expression of DNAs that improve thesynchrony of pollen shed and receptiveness of the female flower parts,i.e., silks, would be of benefit. In addition, expression of genes thatminimize kernel abortion during times of stress would increase theamount of grain to be harvested and hence be of value. Regulation ofcytokinin levels in monocots, such as maize, by introduction andexpression of an isopentenyl transferase gene with appropriateregulatory sequences can improve monocot stress resistance and yield(Gan et al., Science, 270:1986 (1995)).

Given the overall role of water in determining yield, it is contemplatedthat enabling plants to utilize water more efficiently, through theintroduction and expression of novel genes, will improve overallperformance even when soil water availability is not limiting. Byintroducing genes that improve the ability of plants to maximize waterusage across a full range of stresses relating to water availability,yield stability or consistency of yield performance may be realized.

4. Disease Resistance

It is proposed that increased resistance to diseases may be realizedthrough introduction of genes into plants period. It is possible toproduce resistance to diseases caused by viruses, bacteria, fungi, rootpathogens, insects and nematodes. It is also contemplated that controlof mycotoxin producing organisms may be realized through expression ofintroduced genes.

Resistance to viruses may be produced through expression of novel genes.For example, it has been demonstrated that expression of a viral coatprotein in a transgenic plant can impart resistance to infection of theplant by that virus and perhaps other closely related viruses (Cuozzo etal., 1988, Hemenway et al., 1988, Abel et al., 1986). It is contemplatedthat expression of antisense genes targeted at essential viral functionsmay impart resistance to said virus. For example, an antisense genetargeted at the gene responsible for replication of viral nucleic acidmay inhibit said replication and lead to resistance to the virus. It isbelieved that interference with other viral functions through the use ofantisense genes may also increase resistance to viruses. Further it isproposed that it may be possible to achieve resistance to virusesthrough other approaches, including, but not limited to the use ofsatellite viruses.

It is proposed that increased resistance to diseases caused by bacteriaand fungi may be realized through introduction of novel genes. It iscontemplated that genes encoding so-called “peptide antibiotics,”pathogenesis related (PR) proteins, toxin resistance, and proteinsaffecting host-pathogen interactions such as morphologicalcharacteristics will be useful. Peptide antibiotics are polypeptidesequences which are inhibitory to growth of bacteria and othermicroorganisms. For example, the classes of peptides referred to ascecropins and magainins inhibit growth of many species of bacteria andfungi. It is proposed that expression of PR proteins in plants may beuseful in conferring resistance to bacterial disease. These genes areinduced following pathogen attack on a host plant and have been dividedinto at least five classes of proteins (Bol et al., 1990). Includedamongst the PR proteins are beta-1,3-glucanases, chitinases, and osmotinand other proteins that are believed to function in plant resistance todisease organisms. Other genes have been identified that have antifungalproperties, e.g., UDA (stinging nettle lectin) and hevein (Broakgert etal., 1989; Barkai-Golan et al., 1978). It is known that certain plantdiseases are caused by the production of phytotoxins. Resistance tothese diseases could be achieved through expression of a novel gene thatencodes an enzyme capable of degrading or otherwise inactivating thephytotoxin. Expression novel genes that alter the interactions betweenthe host plant and pathogen may be useful in reducing the ability thedisease organism to invade the tissues of the host plant, e.g., anincrease in the waxiness of the leaf cuticle or other morphologicalcharacteristics.

Plant parasitic nematodes are a cause of disease in many plants. It isproposed that it would be possible to make the plant resistant to theseorganisms through the expression of novel genes. It is anticipated thatcontrol of nematode infestations would be accomplished by altering theability of the nematode to recognize or attach to a host plant and/orenabling the plant to produce nematicidal compounds, including but notlimited to proteins.

5. Mycotoxin Reduction/Elimination

Production of mycotoxins, including aflatoxin and fumonisin, by fungiassociated with plants is a significant factor in rendering the grainnot useful. These fungal organisms do not cause disease symptoms and/orinterfere with the growth of the plant, but they produce chemicals(mycotoxins) that are toxic to animals. Inhibition of the growth ofthese fungi would reduce the synthesis of these toxic substances and,therefore, reduce grain losses due to mycotoxin contamination. Novelgenes may be introduced into plants that would inhibit synthesis of themycotoxin without interfering with fungal growth. Expression of a novelgene which encodes an enzyme capable of rendering the mycotoxin nontoxicwould be useful in order to achieve reduced mycotoxin contamination ofgrain. The result of any of the above mechanisms would be a reducedpresence of mycotoxins on grain.

6. Grain Composition or Quality

Genes may be introduced into plants, particularly commercially importantcereals such as maize, wheat or rice, to improve the grain for which thecereal is primarily grown. A wide range of novel transgenic plantsproduced in this manner may be envisioned depending on the particularend use of the grain.

For example, the largest use of maize grain is for feed or food.Introduction of genes that alter the composition of the grain maygreatly enhance the feed or food value. The primary components of maizegrain are starch, protein, and oil. Each of these primary components ofmaize grain may be improved by altering its level or composition.Several examples may be mentioned for illustrative purposes but in noway provide an exhaustive list of possibilities.

The protein of many cereal grains is suboptimal for feed and foodpurposes especially when fed to pigs, poultry, and humans. The proteinis deficient in several amino acids that are essential in the diet ofthese species, requiring the addition of supplements to the grain.Limiting essential amino acids may include lysine, methionine,tryptophan, threonine, valine, arginine, and histidine. Some amino acidsbecome limiting only after the grain is supplemented with other inputsfor feed formulations. For example, when the grain is supplemented withsoybean meal to meet lysine requirements, methionine becomes limiting.The levels of these essential amino acids in seeds and grain may beelevated by mechanisms which include, but are not limited to, theintroduction of genes to increase the biosynthesis of the amino acids,decrease the degradation of the amino acids, increase the storage of theamino acids in proteins, or increase transport of the amino acids to theseeds or grain.

One mechanism for increasing the biosynthesis of the amino acids is tointroduce genes that deregulate the amino acid biosynthetic pathwayssuch that the plant can no longer adequately control the levels that areproduced. This may be done by deregulating or bypassing steps in theamino acid biosynthetic pathway which are normally regulated by levelsof the amino acid end product of the pathway. Examples include theintroduction of genes that encode deregulated versions of the enzymesaspartokinase or dihydrodipicolinic acid (DHDP)-synthase for increasinglysine and threonine production, and anthranilate synthase forincreasing tryptophan production. Reduction of the catabolism of theamino acids may be accomplished by introduction of DNA sequences thatreduce or eliminate the expression of genes encoding enzymes thatcatalyse steps in the catabolic pathways such as the enzymelysine-ketoglutarate reductase.

The protein composition of the grain may be altered to improve thebalance of amino acids in a variety of ways including elevatingexpression of native proteins, decreasing expression of those with poorcomposition, changing the composition of native proteins, or introducinggenes encoding entirely new proteins possessing superior composition.DNA may be introduced that decreases the expression of members of thezein family of storage proteins. This DNA may encode ribozymes orantisense sequences directed to impairing expression of zein proteins orexpression of regulators of zein expression such as the opaque-2 geneproduct. The protein composition of the grain may be modified throughthe phenomenon of cosuppression, i.e., inhibition of expression of anendogenous gene through the expression of an identical structural geneor gene fragment introduced through transformation (Goring et al.,1991). Additionally, the introduced DNA may encode enzymes which degradeseines. The decreases in zein expression that are achieved may beaccompanied by increases in proteins with more desirable amino acidcomposition or increases in other major seed constituents such asstarch. Alternatively, a chimeric gene may be introduced that comprisesa coding sequence for a native protein of adequate amino acidcomposition such as for one of the globulin proteins or 10 kD zein ofmaize and a promoter or other regulatory sequence designed to elevateexpression of said protein. The coding sequence of said gene may includeadditional or replacement codons for essential amino acids. Further, acoding sequence obtained from another species, or, a partially orcompletely synthetic sequence encoding a completely unique peptidesequence designed to enhance the amino acid composition of the seed maybe employed.

The introduction of genes that alter the oil content of the grain may beof value. Increases in oil content may result in increases inmetabolizable energy content and density of the seeds for uses in feedand food. The introduced genes may encode enzymes that remove or reducerate-limitations or regulated steps in fatty acid or lipid biosynthesis.Such genes may include, but are not limited to, those that encodeacetyl-CoA carboxylase, ACP-acyltransferase, beta-ketoacyl-ACP synthase,plus other well known fatty acid biosynthetic activities. Otherpossibilities are genes that encode proteins that do not possessenzymatic activity such as acyl carrier protein. Additional examplesinclude 2-acetyltransferase, oleosin pyruvate dehydrogenase complex,acetyl CoA synthetase, ATP citrate lyase, ADP-glucose pyrophosphorylaseand genes of the carnitine-CoA-acetyl-CoA shuttles. It is anticipatedthat expression of genes related to oil biosynthesis will be targeted tothe plastid, using a plastid transit peptide sequence and preferablyexpressed in the seed embryo. Genes may be introduced that alter thebalance of fatty acids present in the oil providing a more healthful ornutritive feedstuff. The introduced DNA may also encode sequences thatblock expression of enzymes involved in fatty acid biosynthesis,altering the proportions of fatty acids present in the grain such asdescribed below.

Genes may be introduced that enhance the nutritive value of the starchcomponent of the grain, for example by increasing the degree ofbranching, resulting in improved utilization of the starch in cows bydelaying its metabolism.

Besides affecting the major constituents of the grain, genes may beintroduced that affect a variety of other nutritive, processing, orother quality aspects of the grain as used for feed or food. Forexample, pigmentation of the grain may be increased or decreased.Enhancement and stability of yellow pigmentation is desirable in someanimal feeds and may be achieved by introduction of genes that result inenhanced production of xanthophylls and carotenes by eliminatingrate-limiting steps in their production. Such genes may encode alteredforms of the enzymes phytoene synthase, phytoene desaturase, or lycopenesynthase. Alternatively, unpigmented white corn is desirable forproduction of many food products and may be produced by the introductionof DNA which blocks or eliminates steps in pigment production pathways.

Feed or food comprising some cereal grains possesses insufficientquantities of vitamins and must be supplemented to provide adequatenutritive value. Introduction of genes that enhance vitamin biosynthesisin seeds may be envisioned including, for example, vitamins A, E, B₁₂,choline, and the like. For example, maize grain also does not possesssufficient mineral content for optimal nutritive value. Genes thataffect the accumulation or availability of compounds containingphosphorus, sulfur, calcium, manganese, zinc, and iron among otherswould be valuable. An example may be the introduction of a gene thatreduced phytic acid production or encoded the enzyme phytase whichenhances phytic acid breakdown. These genes would increase levels ofavailable phosphate in the diet, reducing the need for supplementationwith mineral phosphate.

Numerous other examples of improvement of cereals for feed and foodpurposes might be described. The improvements may not even necessarilyinvolve the grain, but may, for example, improve the value of the grainfor silage. Introduction of DNA to accomplish this might includesequences that alter lignin production such as those that result in the“brown midrib” phenotype associated with superior feed value for cattle.

In addition to direct improvements in feed or food value, genes may alsobe introduced which improve the processing of grain and improve thevalue of the products resulting from the processing. The primary methodof processing certain grains such as maize is via wetmilling. Maize maybe improved though the expression of novel genes that increase theefficiency and reduce the cost of processing such as by decreasingsteeping time.

Improving the value of wetmilling products may include altering thequantity or quality of starch, oil, corn gluten meal, or the componentsof corn gluten feed. Elevation of starch may be achieved through theidentification and elimination of rate limiting steps in starchbiosynthesis or by decreasing levels of the other components of thegrain resulting in proportional increases in starch. An example of theformer may be the introduction of genes encoding ADP-glucosepyrophosphorylase enzymes with altered regulatory activity or which areexpressed at higher level. Examples of the latter may include selectiveinhibitors of, for example, protein or oil biosynthesis expressed duringlater stages of kernel development.

The properties of starch may be beneficially altered by changing theratio of amylose to amylopectin, the size of the starch molecules, ortheir branching pattern. Through these changes a broad range ofproperties may be modified which include, but are not limited to,changes in gelatinization temperature, heat of gelatinization, clarityof films and pastes, Theological properties, and the like. To accomplishthese changes in properties, genes that encode granule-bound or solublestarch synthase activity or branching enzyme activity may be introducedalone or combination. DNA such as antisense constructs may also be usedto decrease levels of endogenous activity of these enzymes. Theintroduced genes or constructs may possess regulatory sequences thattime their expression to specific intervals in starch biosynthesis andstarch granule development. Furthermore, it may be advisable tointroduce and express genes that result in the in vivo derivatization,or other modification, of the glucose moieties of the starch molecule.The covalent attachment of any molecule may be envisioned, limited onlyby the existence of enzymes that catalyze the derivatizations and theaccessibility of appropriate substrates in the starch granule. Examplesof important derivations may include the addition of functional groupssuch as amines, carboxyls, or phosphate groups which provide sites forsubsequent in vitro derivatizations or affect starch properties throughthe introduction of ionic charges. Examples of other modifications mayinclude direct changes of the glucose units such as loss of hydroxylgroups or their oxidation to aldehyde or carboxyl groups.

Oil is another product of wetmilling of corn and other grains, the valueof which may be improved by introduction and expression of genes. Thequantity of oil that can be extracted by wetmilling may be elevated byapproaches as described for feed and food above. Oil properties may alsobe altered to improve its performance in the production and use ofcooking oil, shortenings, lubricants or other oil-derived products orimprovement of its health attributes when used in the food-relatedapplications. Novel fatty acids may also be synthesized which uponextraction can serve as starting materials for chemical syntheses. Thechanges in oil properties may be achieved by altering the type, level,or lipid arrangement of the fatty acids present in the oil. This in turnmay be accomplished by the addition of genes that encode enzymes thatcatalyze the synthesis of novel fatty acids and the lipids possessingthem or by increasing levels of native fatty acids while possiblyreducing levels of precursors. Alternatively DNA sequences may beintroduced which slow or block steps in fatty acid biosynthesisresulting in the increase in precursor fatty acid intermediates. Genesthat might be added include desaturases, epoxidases, hydratases,dehydratases, and other enzymes that catalyze reactions involving fattyacid intermediates. Representative examples of catalytic steps thatmight be blocked include the desaturations from stearic to oleic acidand oleic to linolenic acid resulting in the respective accumulations ofstearic and oleic acids.

Improvements in the other major cereal wetmilling products, gluten mealand gluten feed, may also be achieved by the introduction of genes toobtain novel plants. Representative possibilities include but are notlimited to those described above for improvement of food and feed value.

In addition it may further be considered that the plant be used for theproduction or manufacturing of useful biological compounds that wereeither not produced at all, or not produced at the same level, in theplant previously. The novel plants producing these compounds are madepossible by the introduction and expression of genes by transformationmethods. The possibilities include, but are not limited to, anybiological compound which is presently produced by any organism such asproteins, nucleic acids, primary and intermediary metabolites,carbohydrate polymers, etc. The compounds may be produced by the plant,extracted upon harvest and/or processing, and used for any presentlyrecognized useful purpose such as pharmaceuticals, fragrances,industrial enzymes to name a few.

Further possibilities to exemplify the range of grain traits orproperties potentially encoded by introduced genes in transgenic plantsinclude grain with less breakage susceptibility for export purposes orlarger grit size when processed by dry milling through introduction ofgenes that enhance gamma-zein synthesis, popcorn with improved poppingquality and expansion volume through genes that increase pericarpthickness, corn with whiter grain for food uses though introduction ofgenes that effectively block expression of enzymes involved in pigmentproduction pathways, and improved quality of alcoholic beverages orsweet corn through introduction of genes which affect flavor such as theshrunken gene (encoding sucrose synthase) for sweet corn.

7. Plant Agronomic Characteristics

Two of the factors determining where plants can be grown are the averagedaily temperature during the growing season and the length of timebetween frosts. Within the areas where it is possible to grow aparticular plant, there are varying limitations on the maximal time itis allowed to grow to maturity and be harvested. The plant to be grownin a particular area is selected for its ability to mature and dry downto harvestable moisture content within the required period of time withmaximum possible yield. Therefore, plant of varying maturities aredeveloped for different growing locations. Apart from the need to drydown sufficiently to permit harvest is the desirability of havingmaximal drying take place in the field to minimize the amount of energyrequired for additional drying post-harvest. Also the more readily thegrain can dry down, the more time there is available for growth andkernel fill. Genes that influence maturity and/or dry down can beidentified and introduced into plant lines using transformationtechniques to create new varieties adapted to different growinglocations or the same growing location but having improved yield tomoisture ratio at harvest. Expression of genes that are involved inregulation of plant development may be especially useful, e.g., theliguleless and rough sheath genes that have been identified in plants.

Genes may be introduced into plants that would improve standability andother plant growth characteristics. For example, expression of novelgenes which confer stronger stalks, improved root systems, or prevent orreduce ear droppage would be of great value to the corn farmer.Introduction and expression of genes that increase the total amount ofphotoassimilate available by, for example, increasing light distributionand/or interception would be advantageous. In addition the expression ofgenes that increase the efficiency of photosynthesis and/or the leafcanopy would further increase gains in productivity. Such approacheswould allow for increased plant populations in the field.

Delay of late season vegetative senescence would increase the flow ofassimilate into the grain and thus increase yield. Overexpression ofgenes within plants that are associated with “stay green” or theexpression of any gene that delays senescence would achieve beadvantageous. For example, a non-yellowing mutant has been identified inFestuca pratensis (Davies et al., 1990). Expression of this gene as wellas others may prevent premature breakdown of chlorophyll and thusmaintain canopy function.

8. Nutrient Utilization

The ability to utilize available nutrients and minerals may be alimiting factor in growth of many plants. It is proposed that it wouldbe possible to alter nutrient uptake, tolerate pH extremes, mobilizationthrough the plant, storage pools, and availability for metabolicactivities by the introduction of novel genes. These modifications wouldallow a plant to more efficiently utilize available nutrients. It iscontemplated that an increase in the activity of, for example, an enzymethat is normally present in the plant and involved in nutrientutilization would increase the availability of a nutrient. An example ofsuch an enzyme would be phytase. It is also contemplated that expressionof a novel gene may make a nutrient source available that was previouslynot accessible, e.g., an enzyme that releases a component of nutrientvalue from a more complex molecule, perhaps a macromolecule.

9. Male Sterility

Male sterility is useful in the production of hybrid seed. It isproposed that male sterility may be produced through expression of novelgenes. For example, it has been shown that expression of genes thatencode proteins that interfere with development of the maleinflorescence and/or gametophyte result in male sterility. Chimericribonuclease genes that express in the anthers of transgenic tobacco andoilseed rape have been demonstrated to lead to male sterility (Marianiet al, 1990).

For example, a number of mutations were discovered in maize that confercytoplasmic male sterility. One mutation in particular, referred to as Tcytoplasm, also correlates with sensitivity to Southern corn leafblight. A DNA sequence, designated TURF-13 (Levings, 1990), wasidentified that correlates with T cytoplasm. It would be possiblethrough the introduction of TURF-13 via transformation to separate malesterility from disease sensitivity. As it is necessary to be able torestore male fertility for breeding purposes and for grain production,it is proposed that genes encoding restoration of male fertility mayalso be introduced.

10. Negative Selectable Markers

Introduction of genes encoding traits that can be selected against maybe useful for eliminating undesirable linked genes. When two or moregenes are introduced together by cotransformation, the genes will belinked together on the host chromosome. For example, a gene encoding aBt gene that confers insect resistance on the plant may be introducedinto a plant together with a bar gene that is useful as a selectablemarker and confers resistance to the herbicide IGNITE® on the plant.However, it may not be desirable to have an insect resistant plant thatis also resistant to the herbicide IGNITE®. It is proposed that onecould also introduce an antisense bar gene that is expressed in thosetissues where one does not want expression of the bar gene, e.g., inwhole plant parts. Hence, although the bar gene is expressed and isuseful as a selectable marker, it is not useful to confer herbicideresistance on the whole plant. The bar antisense gene is a negativeselectable marker. Negative selection is necessary in order to screen apopulation of transformants for rare homologous recombinants generatedthrough gene targeting. For example, a homologous recombinant may beidentified through the inactivation of a gene that was previouslyexpressed in that cell. The antisense gene to neomycinphosphotransferase II (nptII) has been investigated as a negativeselectable marker in tobacco (Nicotiana tabacum) and Arabidopsisthaliana (Xiang and Guerra, 1993). In this example both sense andantisense nptII genes are introduced into a plant through transformationand the resultant plants are sensitive to the antibiotic kanamycin. Anintroduced gene that integrates into the host cell chromosome at thesite of the antisense nptII gene, and inactivates the antisense gene,will make the plant resistant to kanamycin and other aminoglycosideantibiotics. Therefore, rare site specific recombinants may beidentified by screening for antibiotic resistance. Similarly, any gene,native to the plant or introduced through transformation, that wheninactivated confers resistance to a compound, may be useful as anegative selectable marker.

It is contemplated that negative selectable markers may also be usefulin other ways. One application is to construct transgenic lines in whichone could select for transposition to unlinked sites. In the process oftagging it is most common for the transposable element to move to agenetically linked site on the same chromosome. A selectable marker forrecovery of rare plants in which transposition has occurred to anunlinked locus would be useful. For example, the enzyme cytosinedeaminase may be useful for this purpose (Stouggard, 1993). In thepresence of this enzyme the compound 5-fluorocytosine is converted to5-fluoruracil which is toxic to plant and animal cells. If atransposable element is linked to the gene for the enzyme cytosinedeaminase, one may select for transposition to unlinked sites byselecting for transposition events in which the resultant plant is nowresistant to 5-fluorocytosine. The parental plants and plants containingtranspositions to linked sites will remain sensitive to5-fluorocytosine. Resistance to 5-fluorocytosine is due to loss of thecytosine deaminase gene through genetic segregation of the transposableelement and the cytosine deaminase gene. Other genes that encodeproteins that render the plant sensitive to a certain compound will alsobe useful in this context. For example, T-DNA gene 2 from Agrobacteriumtumefaciens encodes a protein that catalyzes the conversion ofalpha-naphthalene acetamide (NAM) to alpha-napthalene acetic acid (NAA)renders plant cells sensitive to high concentrations of NAM (Depicker etal., 1988).

It is also contemplated that negative selectable markers may be usefulin the construction of transposon tagging lines. For example, by markingan autonomous transposable element such as Ac, Master Mu, or En/Spn witha negative selectable marker, one could select for transformants inwhich the autonomous element is not stably integrated into the genome.This would be desirable, for example, when transient expression of theautonomous element is desired to activate in trans the transposition ofa defective transposable element, such as Ds, but stable integration ofthe autonomous element is not desired. The presence of the autonomouselement may not be desired in order to stabilize the defective element,i.e., prevent it from further transposing. However, it is proposed thatif stable integration of an autonomous transposable element is desiredin a plant the presence of a negative selectable marker may make itpossible to eliminate the autonomous element during the breedingprocess.

11. Non-Protein-Expressing Sequences

a. RNA-Expressing

DNA may be introduced into plants for the purpose of expressing RNAtranscripts that function to affect plant phenotype yet are nottranslated into protein. Two examples are antisense RNA and RNA withribozyme activity. Both may serve possible functions in reducing oreliminating expression of native or introduced plant genes.

Genes may be constructed or isolated, which when transcribed, produceantisense RNA that is complementary to all or part(s) of a targetedmessenger RNA(s). The antisense RNA reduces production of thepolypeptide product of the messenger RNA. The polypeptide product may beany protein encoded by the plant genome. The aforementioned genes willbe referred to as antisense genes. An antisense gene may thus beintroduced into a plant by transformation methods to produce a noveltransgenic plant with reduced expression of a selected protein ofinterest. For example, the protein may be an enzyme that catalyzes areaction in the plant. Reduction of the enzyme activity may reduce oreliminate products of the reaction which include any enzymaticallysynthesized compound in the plant such as fatty acids, amino acids,carbohydrates, nucleic acids and the like. Alternatively, the proteinmay be a storage protein, such as a zein, or a structural protein, thedecreased expression of which may lead to changes in seed amino acidcomposition or plant morphological changes respectively. Thepossibilities cited above are provided only by way of example and do notrepresent the full range of applications.

Genes may also be constructed or isolated, which when transcribedproduce RNA enzymes, or ribozymes, which can act as endoribonucleasesand catalyze the cleavage of RNA molecules with selected sequences. Thecleavage of selected messenger RNA's can result in the reducedproduction of their encoded polypeptide products. These genes may beused to prepare novel transgenic plants which possess them. Thetransgenic plants may possess reduced levels of polypeptides includingbut not limited to the polypeptides cited above that may be affected byantisense RNA.

It is also possible that genes may be introduced to produce noveltransgenic plants which have reduced expression of a native gene productby a mechanism of cosuppression. It has been demonstrated in tobacco,tomato, and petunia (Goring et al, 1991; Smith et al., 1990; Napoli etal., 1990; van der Krol et al., 1990) that expression of the sensetranscript of a native gene will reduce or eliminate expression of thenative gene in a manner similar to that observed for antisense genes.The introduced gene may encode all or part of the targeted nativeprotein but its translation may not be required for reduction of levelsof that native protein.

b. Non-RNA-Expressing

For example, DNA elements including those of transposable elements suchas Ds, Ac, or Mu, may be inserted into a gene and cause mutations. TheseDNA elements may be inserted in order to inactivate (or activate) a geneand thereby “tag” a particular trait. In this instance the transposableelement does not cause instability of the tagged mutation, because theutility of the element does not depend on its ability to move in thegenome. Once a desired trait is tagged, the introduced DNA sequence maybe used to clone the corresponding gene, e.g., using the introduced DNAsequence as a PCR primer together with PCR gene cloning techniques(Shapiro, 1983; Dellaporta et al., 1988). Once identified, the entiregene(s) for the particular trait, including control or regulatoryregions where desired may be isolated, cloned and manipulated asdesired. The utility of DNA elements introduced into an organism forpurposed of gene tagging is independent of the DNA sequence and does notdepend on any biological activity of the DNA sequence, i.e.,transcription into RNA or translation into protein. The sole function ofthe DNA element is to disrupt the DNA sequence of a gene.

It is contemplated that unexpressed DNA sequences, including novelsynthetic sequences could be introduced into cells as proprietary“labels” of those cells and plants and seeds thereof. It would not benecessary for a label DNA element to disrupt the function of a geneendogenous to the host organism, as the sole function of this DNA wouldbe to identify the origin of the organism. For example, one couldintroduce a unique DNA sequence into a plant and this DNA element wouldidentify all cells, plants, and progeny of these cells as having arisenfrom that labeled source. It is proposed that inclusion of label DNAswould enable one to distinguish proprietary germplasm or germplasmderived from such, from unlabelled germplasm.

Another possible element which may be introduced is a matrix attachmentregion element (MAR), such as the chicken lysozyme A element (Stief etal., 1989), which can be positioned around an expressible gene ofinterest to effect an increase in overall expression of the gene anddiminish position dependant effects upon incorporation into the plantgenome (Stief et al., 1989; Phi-Van et al., 1990).

Further nucleotide sequences of interest that may be contemplated foruse within the scope of the present invention in operable linkage withthe promoter sequences according to the invention are isolated nucleicacid molecules, e.g., DNA or RNA, comprising a plant nucleotide sequenceaccording to the invention comprising an open reading frame that ispreferentially expressed in a specific tissue, i.e., seed-, root, greentissue (leaf and stem), panicle-, or pollen, or is expressedconstitutively.

B. Marker Genes

In order to improve the ability to identify transformants, one maydesire to employ a selectable or screenable marker gene as, or inaddition to, the expressible gene of interest. “Marker genes” are genesthat impart a distinct phenotype to cells expressing the marker gene andthus allow such transformed cells to be distinguished from cells that donot have the marker. Such genes may encode either a selectable orscreenable marker, depending on whether the marker confers a trait whichone can ‘select’ for by chemical means, i.e., through the use of aselective agent (e.g., a herbicide, antibiotic, or the like), or whetherit is simply a trait that one can identify through observation ortesting, i.e., by ‘screening’ (e.g., the R-locus trait, the greenfluorescent protein (GFP)). Of course, many examples of suitable markergenes are known to the art and can be employed in the practice of theinvention.

Included within the terms selectable or screenable marker genes are alsogenes which encode a “secretable marker” whose secretion can be detectedas a means of identifying or selecting for transformed cells. Examplesinclude markers which encode a secretable antigen that can be identifiedby antibody interaction, or even secretable enzymes which can bedetected by their catalytic activity. Secretable proteins fall into anumber of classes, including small, diffusible proteins detectable,e.g., by ELISA; small active enzymes detectable in extracellularsolution (e.g., alpha-amylase, beta-lactamase, phosphinothricinacetyltransferase); and proteins that are inserted or trapped in thecell wall (e.g., proteins that include a leader sequence such as thatfound in the expression unit of extensin or tobacco PR-S).

With regard to selectable secretable markers, the use of a gene thatencodes a protein that becomes sequestered in the cell wall, and whichprotein includes a unique epitope is considered to be particularlyadvantageous. Such a secreted antigen marker would ideally employ anepitope sequence that would provide low background in plant tissue, apromoter-leader sequence that would impart efficient expression andtargeting across the plasma membrane, and would produce protein that isbound in the cell wall and yet accessible to antibodies. A normallysecreted wall protein modified to include a unique epitope would satisfyall such requirements.

One example of a protein suitable for modification in this manner isextensin, or hydroxyproline rich glycoprotein (HPRG). For example, themaize HPRG (Steifel et al., 1990) molecule is well characterized interms of molecular biology, expression and protein structure. However,any one of a variety of ultilane and/or glycine-rich wall proteins(Keller et al., 1989) could be modified by the addition of an antigenicsite to create a screenable marker.

One exemplary embodiment of a secretable screenable marker concerns theuse of a maize sequence encoding the wall protein HPRG, modified toinclude a 15 residue epitope from the pro-region of murine interleukin,however, virtually any detectable epitope may be employed in suchembodiments, as selected from the extremely wide variety ofantigen-antibody combinations known to those of skill in the art. Theunique extracellular epitope can then be straightforwardly detectedusing antibody labeling in conjunction with chromogenic or fluorescentadjuncts.

Elements of the present disclosure may be exemplified in detail throughthe use of the bar and/or GUS genes, and also through the use of variousother markers. Of course, in light of this disclosure, numerous otherpossible selectable and/or screenable marker genes will be apparent tothose of skill in the art in addition to the one set forth hereinbelow.Therefore, it will be understood that the following discussion isexemplary rather than exhaustive. In light of the techniques disclosedherein and the general recombinant techniques which are known in theart, the present invention renders possible the introduction of anygene, including marker genes, into a recipient cell to generate atransformed plant.

1. Selectable Markers

Possible selectable markers for use in connection with the presentinvention include, but are not limited to, a neo gene (Potrykus et al.,1985) which codes for kanamycin resistance and can be selected for usingkanamycin, G418, paromomycin, and the like; a bar gene which codes forbialaphos or phosphinothricin resistance; a gene which encodes analtered EPSP synthase protein (Hinchee et al., 1988) thus conferringglyphosate resistance; a nitrilase gene such as bxn from Klebsiellaozaenae which confers resistance to bromoxynil (Stalker et al., 1988); amutant acetolactate synthase gene (ALS) which confers resistance toimidazolinone, sulfonylurea or other ALS-inhibiting chemicals (EuropeanPatent Application 154,204, 1985); a methotrexate-resistant DHFR gene(Thillet et al., 1988); a dalapon dehalogenase gene that confersresistance to the herbicide dalapon; a mutated anthranilate synthasegene that confers resistance to 5-methyl tryptophan. Preferredselectable marker genes encode phosphinothricin acetyltransferase;glyphosate resistant EPSPS, aminoglycoside phosphotransferase;hygromycin phosphotransferase, or neomycin phosphotransferase. Where amutant EPSP synthase gene is employed, additional benefit may berealized through the incorporation of a suitable chloroplast transitpeptide, CTP (European Patent Application 0,218,571, 1987).

An illustrative embodiment of a selectable marker gene capable of beingused in systems to select transformants is the genes that encode theenzyme phosphinothricin acetyltransferase, such as the bar gene fromStreptomyces hygroscopicus or the pat gene from Streptomycesviridochromogenes. The enzyme phosphinothricin acetyl transferase (PAT)inactivates the active ingredient in the herbicide bialaphos,phosphinothricin (PPT). PPT inhibits glutamine synthetase, (Murakami etal., 1986; Twell et al., 1989) causing rapid accumulation of ammonia andcell death. The success in using this selective system in conjunctionwith monocots was particularly surprising because of the majordifficulties which have been reported in transformation of cereals(Potrykus, 1989).

Where one desires to employ a bialaphos resistance gene in the practiceof the invention, a particularly useful gene for this purpose is the baror pat genes obtainable from species of Streptomyces (e.g., ATCC No.21,705). The cloning of the bar gene has been described (Murakami etal., 1986; Thompson et al., 1987) as has the use of the bar gene in thecontext of plants other than monocots (De Block et al., 1987; De Blocket al., 1989).

2. Screenable Markers

Screenable markers that may be employed include, but are not limited to,a beta-glucuronidase (GUS) or uidA gene which encodes an enzyme forwhich various chromogenic substrates are known; an R-locus gene, whichencodes a product that regulates the production of anthocyanin pigments(red color) in plant tissues (Dellaporta et al., 1988); a beta-lactamasegene (Sutcliffe, 1978), which encodes an enzyme for which variouschromogenic substrates are known (e.g., PADAC, a chromogeniccephalosporin); a xylE gene (Zukowsky et al., 1983) which encodes acatechol dioxygenase that can convert chromogenic catechols; anV-amylase gene (Ikuta et al., 1990); a tyrosinase gene (Katz et al.,1983) which encodes an enzyme capable of oxidizing tyrosine to DOPA anddopaquinone which in turn condenses to form the easily detectablecompound melanin; a ∃-galactosidase gene, which encodes an enzyme forwhich there are chromogenic substrates; a luciferase (lux) gene (Ow etal., 1986), which allows for bioluminescence detection; or even anaequorin gene (Prasher et al., 1985), which may be employed incalcium-sensitive bioluminescence detection, or a green fluorescentprotein gene (Niedz et al., 1995).

Genes from the maize R gene complex are contemplated to be particularlyuseful as screenable markers. The R gene complex in maize encodes aprotein that acts to regulate the production of anthocyanin pigments inmost seed and plant tissue. A gene from the R gene complex was appliedto maize transformation, because the expression of this gene intransformed cells does not harm the cells. Thus, an R gene introducedinto such cells will cause the expression of a red pigment and, ifstably incorporated, can be visually scored as a red sector. If a maizeline is carries dominant ultila for genes encoding the enzymaticintermediates in the anthocyanin biosynthetic pathway (C2, A1, A2, Bz1and Bz2), but carries a recessive allele at the R locus, transformationof any cell from that line with R will result in red pigment formation.Exemplary lines include Wisconsin 22 which contains the rg-Stadlerallele and TR112, a K55 derivative which is r-g, b, P1. Alternativelyany genotype of maize can be utilized if the C1 and R alleles areintroduced together.

It is further proposed that R gene regulatory regions may be employed inchimeric constructs in order to provide mechanisms for controlling theexpression of chimeric genes. More diversity of phenotypic expression isknown at the R locus than at any other locus (Coe et al., 1988). It iscontemplated that regulatory regions obtained from regions 5′ to thestructural R gene would be valuable in directing the expression ofgenes, e.g., insect resistance, drought resistance, herbicide toleranceor other protein coding regions. For the purposes of the presentinvention, it is believed that any of the various R gene family membersmay be successfully employed (e.g., P, S, Lc, etc.). However, the mostpreferred will generally be Sn (particularly Sn:bol3). Sn is a dominantmember of the R gene complex and is functionally similar to the R and Bloci in that Sn controls the tissue specific deposition of anthocyaninpigments in certain seedling and plant cells, therefore, its phenotypeis similar to R.

A further screenable marker contemplated for use in the presentinvention is firefly luciferase, encoded by the lux gene. The presenceof the lux gene in transformed cells may be detected using, for example,X-ray film, scintillation counting, fluorescent spectrophotometry,low-light video cameras, photon counting cameras or multiwellluminometry. It is also envisioned that this system may be developed forpopulational screening for bioluminescence, such as on tissue cultureplates, or even for whole plant screening. Where use of a screenablemarker gene such as lux or GFP is desired, benefit may be realized bycreating a gene fusion between the screenable marker gene and aselectable marker gene, for example, a GFP-NPTII gene fusion. This couldallow, for example, selection of transformed cells followed by screeningof transgenic plants or seeds.

C. Exemplary DNA Molecules

The invention provides an isolated nucleic acid molecule, e.g., DNA orRNA, comprising a plant nucleotide sequence comprising an open readingframe that is preferentially expressed in a specific plant tissue, i.e.,in seeds, roots, green tissue (leaf and stem), panicles or pollen, or isexpressed constitutively, or a promoter thereof

In one specific embodiment the invention provides an isolated nucleicacid molecule, e.g., DNA or RNA, comprising a plant nucleotide sequencecomprising an open reading frame that is preferentially expressed in aspecific plant tissue, i.e., in seeds, roots, green tissue (leaf andstem), panicles or pollen and which is substantially similar, andpreferably has at least 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90%or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%, nucleicacid sequence identity, to an open reading frame expressed in p1 (i) aseed-specific manner, e.g., one of SEQ ID NOs:1020-1597, 5927, 5940,5941, 5945-5958;

-   -   (ii) a root-specific manner, e.g., one of SEQ ID NOs:801-1019;    -   (iii) a green tissue (leaf and stem)-specific manner, e.g., one        of SEQ ID NOs:399-464;    -   (iv) a panicle-specific manner, e.g., one of SEQ ID NOs:465-720;        or    -   (v) a pollen-specific manner, e.g., one of SEQ ID NOs:721-800;        or the complement thereof.

In another embodiment the invention provides an isolated nucleic acidmolecule, e.g., DNA or RNA, comprising a plant nucleotide sequencecomprising an open reading frame that is constitutively expressed andwhich is substantially similar, and preferably has at least 70%, e.g.,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%,85% 86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, and 99%, nucleic acid sequence identity, to aconstitutively expressed open reading frame, which comprises one of SEQID NOs:1-398 and 5928-5939 or the complement thereof.

In another embodiment, the invention provides an isolated nucleic acidmolecule comprising a promoter which is preferentially expressed in aspecific plant tissue, i.e., in seeds, roots, green tissue (leaf andstem), panicles or pollen and which is substantially similar, andpreferably has at least 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, and even 90%or more, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99%, nucleicacid sequence identity, to a gene comprising a promoter listed in

-   -   (i) SEQ ID NOs:2275-2672, 5959, 5972, 5973, 5977-5990 and 6001        (e.g., including a promoter obtained or obtainable from any one        of SEQ ID NOs:2275-2672, 5959, 5972, 5973, 5977-5990 and 6001)        which directs seed-specific transcription of a linked nucleic        acid segment;    -   (ii) SEQ ID NOs:2144-2274 (e.g., including a promoter obtained        or obtainable from any one of SEQ ID NOs:2144-2274) which        directs root-specific transcription of a linked nucleic acid        segment;    -   (iii) SEQ ID NOs:1886-1918 (e.g., including a promoter obtained        or obtainable from any one of SEQ ID NOs:1886-1918) which        directs green tissue (leaf and stem)-specific transcription of a        linked nucleic acid segment;    -   (iv) SEQ ID NOs:1919-2085 (e.g., including a promoter obtained        or obtainable from any one of SEQ ID NOs:1919-2085) which        directs panicle-specific transcription of a linked nucleic acid        segment;    -   (v) SEQ ID NOs:2086-2143 (e.g., including a promoter obtained or        obtainable from any one of SEQ ID NOs:2086-2143) which directs        pollen-specific transcription of a linked nucleic acid segment.

In yet another embodiment, the invention provides an isolated nucleicacid molecule comprising a promoter constitutively expressed and whichis substantially similar, and preferably has at least 70%, e.g., 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, and even 90% or more, e.g., 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, and 99%, nucleic acid sequence identity, to a genecomprising a promoter listed in

-   -   (vi) SEQ ID NOs:1598-1885 and 5960-5971 (e.g., including a        promoter obtained or obtainable from any one of SEQ ID        NOs:1598-1885 and 5960-5971, respectively) which directs        constitutve transcription of a linked nucleic acid segment.

The present invention further provides a composition, an expressioncassette or a recombinant vector containing the nucleic acid molecule ofthe invention, and host cells comprising the expression cassette orvector, e.g., comprising a plasmid. In particular, the present inventionprovides an expression cassette or a recombinant vector comprising apromoter linked to a nucleic acid segment comprising an open readingframe according to the invention which, when present in a plant, plantcell or plant tissue, results in transcription of the linked nucleicacid segment. Further, the invention provides isolated polypeptidesencoded by any one of the open reading frames comprising SEQ IDNOs:1-1597, 5927, 5940, 5941, 5945-5958, or the orthologs thereof, e.g.,an open reading frame comprising one of SEQ ID NOs:2673-5926.

The choice of promoter directing expression of a nucleic acid segmentcomprising an open reading frame according to the invention will varydepending on the temporal and spatial requirements for expression, andalso depending on the target species. In some cases, expression inmultiple tissues is desirable. While in others, tissue-specific, e.g.,seed-specific, expression is desirable. Although many promoters fromdicotyledons have been shown to be operational in monocotyledons andvice versa, ideally dicotyledonous promoters are selected for expressionin dicotyledons, and monocotyledonous promoters for expression inmonocotyledons. However, there is no restriction to the provenance ofselected promoters; it is sufficient that they are operational indriving the expression of the nucleotide sequences in the desired cell.

These promoters include, but are not limited to, constitutive,inducible, temporally regulated, developmentally regulated,spatially-regulated, chemically regulated, stress-responsive,tissue-specific, viral and synthetic promoters. Promoter sequences areknown to be strong or weak. A strong promoter provides for a high levelof gene expression, whereas a weak promoter provides for a very lowlevel of gene expression. An inducible promoter is a promoter thatprovides for the turning on and off of gene expression in response to anexogenously added agent, or to an environmental or developmentalstimulus. A bacterial promoter such as the P_(tac) promoter can beinduced to varying levels of gene expression depending on the level ofisothiopropylgalactoside added to the transformed bacterial cells. Anisolated promoter sequence that is a strong promoter for heterologousnucleic acid is advantageous because it provides for a sufficient levelof gene expression to allow for easy detection and selection oftransformed cells and provides for a high level of gene expression whendesired.

Within a plant promoter region there are several domains that arenecessary for full function of the promoter. The first of these domainslies immediately upstream of the structural gene and forms the “corepromoter region” containing consensus sequences, normally 70 base pairsimmediately upstream of the gene. The core promoter region contains thecharacteristic CAAT and TATA boxes plus surrounding sequences, andrepresents a transcription initiation sequence that defines thetranscription start point for the structural gene.

The presence of the core promoter region defines a sequence as being apromoter: if the region is absent, the promoter is non-functional.Furthermore, the core promoter region is insufficient to provide fullpromoter activity. A series of regulatory sequences upstream of the coreconstitute the remainder of the promoter. The regulatory sequencesdetermine expression level, the spatial and temporal pattern ofexpression and, for an important subset of promoters, expression underinductive conditions (regulation by external factors such as light,temperature, chemicals, hormones).

A range of naturally-occurring promoters are known to be operative inplants and have been used to drive the expression of heterologous (bothforeign and endogenous) genes in plants: for example, the constitutive35S cauliflower mosaic virus (CaMV) promoter, the ripening-enhancedtomato polygalacturonase promoter (Bird et al., 1988), the E8 promoter(Diekman & Fischer, 1988) and the fruit specific 2A1 promoter (Pear etal., 1989) and many others, e.g., U2 and U5 snRNA promoters from maize,the promoter from alcohol dehydrogenase, the Z4 promoter from a geneencoding the Z4 22 kD zein protein, the Z10 promoter from a geneencoding a 10 kD zein protein, a Z27 promoter from a gene encoding a 27kD zein protein, the A20 promoter from the gene encoding a 19 kD -zeinprotein, inducible promoters, such as the light inducible promoterderived from the pea rbcS gene and the actin promoter from rice, e.g.,the actin 2 promoter (WO 00/70067); seed specific promoters, such as thephaseolin promoter from beans, may also be used. The nucleotidesequences of this invention can also be expressed under the regulationof promoters that are chemically regulated. This enables the nucleicacid sequence or encoded polypeptide to be synthesized only when thecrop plants are treated with the inducing chemicals. Chemical inductionof gene expression is detailed in EP 0 332 104 (to Ciba-Geigy) and U.S.Pat. No. 5,614,395. A preferred promoter for chemical induction is thetobacco PR-la promoter.

Examples of some constitutive promoters which have been describedinclude the rice actin 1 (Wang et al., 1992; U.S. Pat. No. 5,641,876),CaMV 35S (Odell et al., 1985), CaMV 19S (Lawton et al., 1987), nos, Adh,sucrose synthase; and the ubiquitin promoters.

Examples of tissue specific promoters which have been described includethe lectin (Vodkin, 1983; Lindstrom et al., 1990) corn alcoholdehydrogenase 1 (Vogel et al., 1989; Dennis et al., 1984), corn lightharvesting complex (Simpson, 1986; Bansal et al., 1992), corn heat shockprotein (Odell et al., 1985), pea small subunit RuBP carboxylase(Poulsen et al., 1986), Ti plasmid mannopine synthase (Langridge et al.,1989), Ti plasmid nopaline synthase (Langridge et al., 1989), petuniachalcone isomerase (vanTunen et al., 1988), bean glycine rich protein 1(Keller et al., 1989), truncated CaMV 35s (Odell et al., 1985), potatopatatin (Wenzler et al., 1989), root cell (Yamamoto et al., 1990), maizezein (Reina et al., 1990; Kriz et al., 1987; Wandelt et al., 1989;Langridge et al., 1983; Reina et al., 1990), globulin-1 (Belanger etal., 1991), α-tubulin, cab (Sullivan et al., 1989), PEPCase (Hudspeth &Grula, 1989), R gene complex-associated promoters (Chandler et al.,1989), histone, and chalcone synthase promoters (Franken et al., 1991).Tissue specific enhancers are described in Fromm et al. (1989).

Inducible promoters that have been described include the ABA- andturgor-inducible promoters, the promoter of the auxin-binding proteingene (Schwob et al., 1993), the UDP glucose flavonoidglycosyl-transferase gene promoter (Ralston et al., 1988), the MPIproteinase inhibitor promoter (Cordero et al., 1994), and theglyceraldehyde-3-phosphate dehydrogenase gene promoter (Kohler et al.,1995; Quigley et al., 1989; Martinez et al., 1989).

Several other tissue-specific regulated genes and/or promoters have beenreported in plants. These include genes encoding the seed storageproteins (such as napin, cruciferin, beta-conglycinin, and phaseolin)zein or oil body proteins (such as oleosin), or genes involved in fattyacid biosynthesis (including acyl carrier protein, stearoyl-ACPdesaturase. And fatty acid desaturases (fad 2-1)), and other genesexpressed during embryo development (such as Bce4, see, for example, EP255378 and Kridl et al., 1991). Particularly useful for seed-specificexpression is the pea vicilin promoter (Czako et al., 1992). (See alsoU.S. Pat. No. 5,625,136, herein incorporated by reference.) Other usefulpromoters for expression in mature leaves are those that are switched onat the onset of senescence, such as the SAG promoter from Arabidopsis(Gan et al., 1995).

A class of fruit-specific promoters expressed at or during antithesisthrough fruit development, at least until the beginning of ripening, isdiscussed in U.S. Pat. No. 4,943,674. cDNA clones that arepreferentially expressed in cotton fiber have been isolated (John etal., 1992). cDNA clones from tomato displaying differential expressionduring fruit development have been isolated and characterized (Manssonet al., 1985, Slater et al., 1985). The promoter for polygalacturonasegene is active in fruit ripening. The polygalacturonase gene isdescribed in U.S. Pat. No. 4,535,060, U.S. Pat. No. 4,769,061, U.S. Pat.No. 4,801,590, and U.S. Pat. No. 5,107,065, which disclosures areincorporated herein by reference.

Other examples of tissue-specific promoters include those that directexpression in leaf cells following damage to the leaf (for example, fromchewing insects), in tubers (for example, patatin gene promoter), and infiber cells (an example of a developmentally-regulated fiber cellprotein is E6 (John et al., 1992). The E6 gene is most active in fiber,although low levels of transcripts are found in leaf, ovule and flower.

The tissue-specificity of some “tissue-specific” promoters may not beabsolute and may be tested by one skilled in the art using thediphtheria toxin sequence. One can also achieve tissue-specificexpression with “leaky” expression by a combination of differenttissue-specific promoters (Beals et al., 1997). Other tissue-specificpromoters can be isolated by one skilled in the art (see U.S. Pat. No.5,589,379). Several inducible promoters (“gene switches”) have beenreported. Many are described in the review by Gatz (1996) and Gatz(1997). These include tetracycline repressor system, Lac repressorsystem, copper-inducible systems, salicylate-inducible systems (such asthe PR1a system), glucocorticoid- (Aoyama et al., 1997) andecdysome-inducible systems. Also included are the benzene sulphonamide-(U.S. Pat. No. 5,364,780) and alcohol-(WO 97/06269 and WO 97/06268)inducible systems and glutathione S-transferase promoters. Other studieshave focused on genes inducibly regulated in response to environmentalstress or stimuli such as increased salinity. Drought, pathogen andwounding. (Graham et al., 1985; Graham et al., 1985, Smith et al.,1986). Accumulation of metallocarboxypeptidase-inhibitor protein hasbeen reported in leaves of wounded potato plants (Graham et al., 1981).Other plant genes have been reported to be induced methyl jasmonate,elicitors, heat-shock, anaerobic stress, or herbicide safeners.

Regulated expression of the chimeric transacting viral replicationprotein can be further regulated by other genetic strategies. Forexample, Cre-mediated gene activation as described by Odell et al. 1990.Thus, a DNA fragment containing 3′ regulatory sequence bound by loxsites between the promoter and the replication protein coding sequencethat blocks the expression of a chimeric replication gene from thepromoter can be removed by Cre-mediated excision and result in theexpression of the trans-acting replication gene. In this case, thechimeric Cre gene, the chimeric trans-acting replication gene, or bothcan be under the control of tissue- and developmental-specific orinducible promoters. An alternate genetic strategy is the use of tRNAsuppressor gene. For example, the regulated expression of a tRNAsuppressor gene can conditionally control expression of a trans-actingreplication protein coding sequence containing an appropriatetermination codon as described by Ulmasov et al. 1997. Again, either thechimeric tRNA suppressor gene, the chimeric transacting replicationgene, or both can be under the control of tissue- anddevelopmental-specific or inducible promoters.

Frequently it is desirable to have continuous or inducible expression ofa DNA sequence throughout the cells of an organism in atissue-independent manner. For example, increased resistance of a plantto infection by soil- and airborne-pathogens might be accomplished bygenetic manipulation of the plant's genome to comprise a continuouspromoter operably linked to a heterologous pathogen-resistance gene suchthat pathogen-resistance proteins are continuously expressed throughoutthe plant's tissues.

Alternatively, it might be desirable to inhibit expression of a nativeDNA sequence within a plant's tissues to achieve a desired phenotype. Inthis case, such inhibition might be accomplished with transformation ofthe plant to comprise a constitutive, tissue-independent promoteroperably linked to an antisense nucleotide sequence, such thatconstitutive expression of the antisense sequence produces an RNAtranscript that interferes with translation of the mRNA of the nativeDNA sequence.

To define a minimal promoter region, a DNA segment representing thepromoter region is removed from the 5′ region of the gene of interestand operably linked to the coding sequence of a marker (reporter) geneby recombinant DNA techniques well known to the art. The reporter geneis operably linked downstream of the promoter, so that transcriptsinitiating at the promoter proceed through the reporter gene. Reportergenes generally encode proteins which are easily measured, including,but not limited to, chloramphenicol acetyl transferase (CAT),beta-glucuronidase (GUS), green fluorescent protein (GFP),beta-galactosidase (beta-GAL), and luciferase.

The construct containing the reporter gene under the control of thepromoter is then introduced into an appropriate cell type bytransfection techniques well known to the art. To assay for the reporterprotein, cell lysates are prepared and appropriate assays, which arewell known in the art, for the reporter protein are performed. Forexample, if CAT were the reporter gene of choice, the lysates from cellstransfected with constructs containing CAT under the control of apromoter under study are mixed with isotopically labeled chloramphenicoland acetyl-coenzyme A (acetyl-CoA). The CAT enzyme transfers the acetylgroup from acetyl-CoA to the 2- or 3-position of chloramphenicol. Thereaction is monitored by thin-layer chromatography, which separatesacetylated chloramphenicol from unreacted material. The reactionproducts are then visualized by autoradiography.

The level of enzyme activity corresponds to the amount of enzyme thatwas made, which in turn reveals the level of expression from thepromoter of interest. This level of expression can be compared to otherpromoters to determine the relative strength of the promoter understudy. In order to be sure that the level of expression is determined bythe promoter, rather than by the stability of the mRNA, the level of thereporter mRNA can be measured directly, such as by Northern blotanalysis.

Once activity is detected, mutational and/or deletional analyses may beemployed to determine the minimal region and/or sequences required toinitiate transcription. Thus, sequences can be deleted at the 5′ end ofthe promoter region and/or at the 3′ end of the promoter region, andnucleotide substitutions introduced. These constructs are thenintroduced to cells and their activity determined.

In one embodiment, the promoter may be a gamma zein promoter, an oleosinole 16 promoter, a globulinI promoter, an actin I promoter, an actin clpromoter, a sucrose synthetase promoter, an INOPS promoter, an EXM5promoter, a globulin2 promoter, a b-32, ADPG-pyrophosphorylase promoter,an LtpI promoter, an Ltp2 promoter, an oleosin ole17 promoter, anoleosin ole18 promoter, an actin 2 promoter, a pollen-specific proteinpromoter, a pollen-specific pectate lyase promoter, an anther-specificprotein promoter, an anther-specific gene RTS2 promoter, apollen-specific gene promoter, a tapetum-specific gene promoter,tapetum-specific gene RAB24 promoter, a anthranilate synthase alphasubunit promoter, an alpha zein promoter, an anthranilate synthase betasubunit promoter, a dihydrodipicolinate synthase promoter, a Thi1promoter, an alcohol dehydrogenase promoter, a cab binding proteinpromoter, an H3C4 promoter, a RUBISCO SS starch branching enzymepromoter, an ACCase promoter, an actin3 promoter, an actin7 promoter, aregulatory protein GF14-12 promoter, a ribosomal protein L9 promoter, acellulose biosynthetic enzyme promoter, an S-adenosyl-L-homocysteinehydrolase promoter, a superoxide dismutase promoter, a C-kinase receptorpromoter, a phosphoglycerate mutase promoter, a root-specific RCc3 mRNApromoter, a glucose-6 phosphate isomerase promoter, apyrophosphate-fructose 6-phosphatelphosphotransferase promoter, anubiquitin promoter, a beta-ketoacyl-ACP synthase promoter, a 33 kDaphotosystem 11 promoter, an oxygen evolving protein promoter, a 69 kDavacuolar ATPase subunit promoter, a metallothionein-like proteinpromoter, a glyceraldehyde-3-phosphate dehydrogenase promoter, an ABA-and ripening-inducible-like protein promoter, a phenylalanine ammonialyase promoter, an adenosine triphosphatase S-adenosyl-L-homocysteinehydrolase promoter, an α-tubulin promoter, a cab promoter, a PEPCasepromoter, an R gene promoter, a lectin promoter, a light harvestingcomplex promoter, a heat shock protein promoter, a chalcone synthasepromoter, a zein promoter, a globulin-1 promoter, an ABA promoter, anauxin-binding protein promoter, a UDP glucose flavonoidglycosyl-transferase gene promoter, an NTI promoter, an actin promoter,an opaque 2 promoter, a b70 promoter, an oleosin promoter, a CaMV 35Spromoter, a CaMV 19S promoter, a histone promoter, a turgor-induciblepromoter, a pea small subunit RuBP carboxylase promoter, a Ti plasmidmannopine synthase promoter, Ti plasmid nopaline synthase promoter, apetunia chalcone isomerase promoter, a bean glycine rich protein Ipromoter, a CaMV 35S transcript promoter, a potato patatin promoter, ora S-E9 small subunit RuBP carboxylase promoter.

III. Transformed (Transgenic) Plants of the Invention and Methods ofPreparation

Plant species may be transformed with the DNA construct of the presentinvention by the DNA-mediated transformation of plant cell protoplastsand subsequent regeneration of the plant from the transformedprotoplasts in accordance with procedures well known in the art.

Any plant tissue capable of subsequent clonal propagation, whether byorganogenesis or embryogenesis, may be transformed with a vector of thepresent invention. The term “organogenesis,” as used herein, means aprocess by which shoots and roots are developed sequentially frommeristematic centers; the term “embryogenesis,” as used herein, means aprocess by which shoots and roots develop together in a concertedfashion (not sequentially), whether from somatic cells or gametes. Theparticular tissue chosen will vary depending on the clonal propagationsystems available for, and best suited to, the particular species beingtransformed. Exemplary tissue targets include leaf disks, pollen,embryos, cotyledons, hypocotyls, megagametophytes, callus tissue,existing meristematic tissue (e.g., apical meristems, axillary buds, androot meristems), and induced meristem tissue (e.g., cotyledon meristemand ultilane meristem).

Plants of the present invention may take a variety of forms. The plantsmay be chimeras of transformed cells and non-transformed cells; theplants may be clonal transformants (e.g., all cells transformed tocontain the expression cassette); the plants may comprise grafts oftransformed and untransformed tissues (e.g., a transformed root stockgrafted to an untransformed scion in citrus species). The transformedplants may be propagated by a variety of means, such as by clonalpropagation or classical breeding techniques. For example, firstgeneration (or T1) transformed plants may be selfed to give homozygoussecond generation (or T2) transformed plants, and the T2 plants furtherpropagated through classical breeding techniques. A dominant selectablemarker (such as npt II) can be associated with the expression cassetteto assist in breeding.

Thus, the present invention provides a transformed (transgenic) plantcell, in planta or ex planta, including a transformed plastid or otherorganelle, e.g., nucleus, mitochondria or chloroplast. The presentinvention may be used for transformation of any plant species,including, but not limited to, cells from corn (Zea mays), Brassica sp.(e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracanal)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Cofea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea ultilane), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, duckweed(Lemna), barley, vegetables, ornamentals, and conifers.

Duckweed (Lemna, see WO 00/07210) includes members of the familyLemnaceae. There are known four genera and 34 species of duckweed asfollows: genus Lemna (L. aequinoctialis, L. disperma, L. ecuadoriensis,L. gibba, L. japonica, L. minor, L. miniscula, L. obscura, L.perpusilla, L. tenera, L. trisulca, L. turionifera, L. valdiviana);genus Spirodela (S. intermedia, S. polyrrhiza, S. punctata); genusWoffia (Wa. Angusta, Wa. Arrhiza, Wa. Australina, Wa. Borealis, Wa.Brasiliensis, Wa. Columbiana, Wa. Elongata, Wa. Globosa, Wa.Microscopica, Wa. Neglecta) and genus Wofiella ultila, Wl. ultilanen,Wl. gladiata, Wl. ultila, WI. lingulata, Wl. repunda, Wl. rotunda, andWl. neotropica). Any other genera or species of Lemnaceae, if theyexist, are also aspects of the present invention. Lemna gibba, Lemnaminor, and Lemna miniscula are preferred, with Lemna minor and Lemnaminiscula being most preferred. Lemna species can be classified usingthe taxonomic scheme described by Landolt, Biosystematic Investigationon the Family of Duckweeds: The family of Lemnaceae—A Monograph Study.Geobatanischen Institut ETH, Stiftung Rubel, Zurich (1986)).

Vegetables within the scope of the invention include tomatoes(Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans(Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrusspp.), and members of the genus Cucumis such as cucumber (C. sativus),cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentalsinclude azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea),hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipaspp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation(Dianthus caryophyllus), poinsettia (Euphorbia pukherrima), andchrysanthemum. Conifers that may be employed in practicing the presentinvention include, for example, pines such as loblolly pine (Pinustaeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosa),lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata),Douglas-fir (Pseudotsuga menziesii); Western hemlock (Tsuga ultilane);Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firssuch as silver fir (Abies amabilis) and balsam fir (Abies balsamea); andcedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis). Leguminous plants include beans and peas.Beans include guar, locust bean, fenugreek, soybean, garden beans,cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc. Legumesinclude, but are not limited to, Arachis, e.g., peanuts, Vicia, e.g.,crown vetch, hairy vetch, adzuki bean, mung bean, and chickpea, Lupinus,e.g., lupine, trifolium, Phaseolus, e.g., common bean and lima bean,Pisum, e.g., field bean, Melilotus, e.g., clover, Medicago, e.g.,alfalfa, Lotus, e.g., trefoil, lens, e.g., lentil, and false indigo.Preferred forage and turf grass for use in the methods of the inventioninclude alfalfa, orchard grass, tall fescue, perennial ryegrass,creeping bent grass, and redtop.

Other plants within the scope of the invention include Acacia, aneth,artichoke, arugula, blackberry, canola, cilantro, clementines, escarole,eucalyptus, fennel, grapefruit, honey dew, jicama, kiwifruit, lemon,lime, mushroom, nut, okra, orange, parsley, persimmon, plantain,pomegranate, poplar, radiata pine, radicchio, Southern pine, sweetgum,tangerine, triticale, vine, yams, apple, pear, quince, cherry, apricot,melon, hemp, buckwheat, grape, raspberry, chenopodium, blueberry,nectarine, peach, plum, strawberry, watermelon, eggplant, pepper,cauliflower, Brassica, e.g., broccoli, cabbage, ultilan sprouts, onion,carrot, leek, beet, broad bean, celery, radish, pumpkin, endive, gourd,garlic, snapbean, spinach, squash, turnip, ultilane, and zucchini.

Ornamental plants within the scope of the invention include impatiens,Begonia, Pelargonium, Viola, Cyclamen, Verbena, Vinca, Tagetes, Primula,Saint Paulia, Agertum, Amaranthus, Antihirrhinum, Aquilegia, Cineraria,Clover, Cosmo, Cowpea, Dahlia, Datura, Delphinium, Gerbera, Gladiolus,Gloxinia, Hippeastrum, Mesembryanthemum, Salpiglossos, and Zinnia. Otherplants within the scope of the invention are shown in the Tablehereinabove.

Preferably, transgenic plants of the present invention are crop plantsand in particular cereals (for example, corn, alfalfa, sunflower, rice,Brassica, canola, soybean, barley, soybean, sugarbeet, cotton,safflower, peanut, sorghum, wheat, millet, tobacco, etc.), and even morepreferably corn, rice and soybean.

Transformation of plants can be undertaken with a single DNA molecule ormultiple DNA molecules (i.e., co-transformation), and both thesetechniques are suitable for use with the expression cassettes of thepresent invention. Numerous transformation vectors are available forplant transformation, and the expression cassettes of this invention canbe used in conjunction with any such vectors. The selection of vectorwill depend upon the preferred transformation technique and the targetspecies for transformation.

A variety of techniques are available and known to those skilled in theart for introduction of constructs into a plant cell host. Thesetechniques generally include transformation with DNA employing A.tumefaciens or A. rhizogenes as the transforming agent, liposomes, PEGprecipitation, electroporation, DNA injection, direct DNA uptake,microprojectile bombardment, particle acceleration, and the like (See,for example, EP 295959 and EP 138341) (see below). However, cells otherthan plant cells may be transformed with the expression cassettes of theinvention. The general descriptions of plant expression vectors andreporter genes, and Agrobacterium and Agrobacterium-mediated genetransfer, can be found in Gruber et al. (1993).

Expression vectors containing genomic or synthetic fragments can beintroduced into protoplasts or into intact tissues or isolated cells.Preferably expression vectors are introduced into intact tissue. Generalmethods of culturing plant tissues are provided for example by Maki etal., (1993); and by Phillips et al. (1988). Preferably, expressionvectors are introduced into maize or other plant tissues using a directgene transfer method such as microprojectile-mediated delivery, DNAinjection, electroporation and the like. More preferably expressionvectors are introduced into plant tissues using the microprojectilemedia delivery with the biolistic device. See, for example, Tomes et al.(1995). The vectors of the invention can not only be used for expressionof structural genes but may also be used in exon-trap cloning, orpromoter trap procedures to detect differential gene expression invarieties of tissues, (Lindsey et al., 1993; Auch & Reth et al.).

It is particularly preferred to use the binary type vectors of Ti and Riplasmids of Agrobacterium spp. Ti-derived vectors transform a widevariety of higher plants, including monocotyledonous and dicotyledonousplants, such as soybean, cotton, rape, tobacco, and rice (Pacciotti etal., 1985: Byrne et al., 1987; Sukhapinda et al., 1987; Lorz et al.,1985; Potrykus, 1985; Park et al., 1985: Hiei et al., 1994). The use ofT-DNA to transform plant cells has received extensive study and is amplydescribed (EP 120516; Hoekema, 1985; Knauf, et al., 1983; and An et al.,1985). For introduction into plants, the chimeric genes of the inventioncan be inserted into binary vectors as described in the examples.

Other transformation methods are available to those skilled in the art,such as direct uptake of foreign DNA constructs (see EP 295959),techniques of electroporation (Fromm et al., 1986) or high velocityballistic bombardment with metal particles coated with the nucleic acidconstructs (Kline et al., 1987, and U.S. Pat. No. 4,945,050). Oncetransformed, the cells can be regenerated by those skilled in the art.Of particular relevance are the recently described methods to transformforeign genes into commercially important crops, such as rapeseed (DeBlock et al., 1989), sunflower (Everett et al., 1987), soybean (McCabeet al., 1988; Hinchee et al., 1988; Chee et al., 1989; Christou et al.,1989; EP 301749), rice (Hiei et al., 1994), and corn (Gordon Kamm etal., 1990; Fromm et al., 1990).

Those skilled in the art will appreciate that the choice of method mightdepend on the type of plant, i.e., monocotyledonous or dicotyledonous,targeted for transformation. Suitable methods of transforming plantcells include, but are not limited to, microinjection (Crossway et al.,1986), electroporation (Riggs et al., 1986), Agrobacterium-mediatedtransformation (Hinchee et al., 1988), direct gene transfer (Paszkowskiet al., 1984), and ballistic particle acceleration using devicesavailable from Agracetus, Inc., Madison, Wis. And BioRad, Hercules,Calif. (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; andMcCabe et al., 1988). Also see, Weissinger et al., 1988; Sanford et al.,1987 (onion); Christou et al., 1988 (soybean); McCabe et al., 1988(soybean); Datta et al., 1990 (rice); Klein et al., 1988 (maize); Kleinet al., 1988 (maize); Klein et al., 1988 (maize); Fromm et al., 1990(maize); and Gordon-Kamm et al., 1990 (maize); Svab et al., 1990(tobacco chloroplast); Koziel et al., 1993 (maize); Shimamoto et al.,1989 (rice); Christou et al., 1991 (rice); European Patent ApplicationEP 0 332 581 (orchardgrass and other Pooideae); Vasil et al., 1993(wheat); Weeks et al., 1993 (wheat). In one embodiment, the protoplasttransformation method for maize is employed (European Patent ApplicationEP 0 292 435, U.S. Pat. No. 5,350,689).

In another embodiment, a nucleotide sequence of the present invention isdirectly transformed into the plastid genome. Plastid transformationtechnology is extensively described in U.S. Pat. Nos. 5,451,513,5,545,817, and 5,545,818, in PCT application no. WO 95/16783, and inMcBride et al., 1994. The basic technique for chloroplast transformationinvolves introducing regions of cloned plastid DNA flanking a selectablemarker together with the gene of interest into a suitable target tissue,e.g., using biolistics or protoplast transformation (e.g., calciumchloride or PEG mediated transformation). The 1 to 1.5 kb flankingregions, termed targeting sequences, facilitate orthologousrecombination with the plastid genome and thus allow the replacement ormodification of specific regions of the plastome. Initially, pointmutations in the chloroplast 16S rRNA and rps12 genes conferringresistance to spectinomycin and/or streptomycin are utilized asselectable markers for transformation (Svab et al., 1990; Staub et al.,1992). This resulted in stable homoplasmic transformants at a frequencyof approximately one per 100 bombardments of target leaves. The presenceof cloning sites between these markers allowed creation of a plastidtargeting vector for introduction of foreign genes (Staub et al., 1993).Substantial increases in transformation frequency are obtained byreplacement of the recessive rRNA or r-protein antibiotic resistancegenes with a dominant selectable marker, the bacterial aadA geneencoding the spectinomycin-detoxifying enzymeaminoglycoside-3N-adenyltransferase (Svab et al., 1993). Otherselectable markers useful for plastid transformation are known in theart and encompassed within the scope of the invention. Typically,approximately 15-20 cell division cycles following transformation arerequired to reach a homoplastidic state. Plastid expression, in whichgenes are inserted by orthologous recombination into all of the severalthousand copies of the circular plastid genome present in each plantcell, takes advantage of the enormous copy number advantage overnuclear-expressed genes to permit expression levels that can readilyexceed 10% of the total soluble plant protein. In a preferredembodiment, a nucleotide sequence of the present invention is insertedinto a plastid targeting vector and transformed into the plastid genomeof a desired plant host. Plants homoplastic for plastid genomescontaining a nucleotide sequence of the present invention are obtained,and are preferentially capable of high expression of the nucleotidesequence.

Agrobacterium tumefaciens cells containing a vector comprising anexpression cassette of the present invention, wherein the vectorcomprises a Ti plasmid, are useful in methods of making transformedplants. Plant cells are infected with an Agrobacterium tumefaciens asdescribed above to produce a transformed plant cell, and then a plant isregenerated from the transformed plant cell. Numerous Agrobacteriumvector systems useful in carrying out the present invention are known.

For example, vectors are available for transformation usingAgrobacterium tumefaciens. These typically carry at least one T-DNAborder sequence and include vectors such as pBIN19 (Bevan, 1984). In onepreferred embodiment, the expression cassettes of the present inventionmay be inserted into either of the binary vectors pCIB200 and pCIB2001for use with Agrobacterium. These vector cassettes forAgrobacterium-mediated transformation wear constructed in the followingmanner. PTJS75kan was created by Nan digestion of pTJS75 (Schmidhauser &Helinski, 1985) allowing excision of the tetracycline-resistance gene,followed by insertion of an AccI fragment from pUC4K carrying an NPTII(Messing & Vierra, 1982; Bevan et al., 1983; McBride et al., 1990). XhoIlinkers were ligated to the EcoRV fragment of pCIB7 which contains theleft and right T-DNA borders, a plant selectable nos/nptII chimeric geneand the pUC polylinker (Rothstein et al., 1987), and the XhoI-digestedfragment was cloned into SalI-digested pTJS75kan to create pCIB200 (seealso EP 0 332 104, example 19). PCIB200 contains the following uniquepolylinker restriction sites: EcoRI, SstI, KpnI, BglII, XbaI, and SalI.The plasmid pCIB2001 is a derivative of pCIB200 which was created by theinsertion into the polylinker of additional restriction sites. Uniquerestriction sites in the polylinker of pCIB2001 are EcoRI, SstI, KpnI,BglII, XbaI, SalI, MluI, BclI, AvrII, ApaI, HpaI, and StuI. PCIB2001, inaddition to containing these unique restriction sites also has plant andbacterial kanamycin selection, left and right T-DNA borders forAgrobacterium-mediated transformation, the RK2-derived trfA function formobilization between E. coli and other hosts, and the OriT and OriVfunctions also from RK2. The pCIB2001 polylinker is suitable for thecloning of plant expression cassettes containing their own regulatorysignals.

An additional vector useful for Agrobacterium-mediated transformation isthe binary vector pCIB 10, which contains a gene encoding kanamycinresistance for selection in plants, T-DNA right and left bordersequences and incorporates sequences from the wide host-range plasmidpRK252 allowing it to replicate in both E. coli and Agrobacterium. Itsconstruction is described by Rothstein et al., 1987. Various derivativesof pCIB10 have been constructed which incorporate the gene forhygromycin B phosphotransferase described by Gritz et al., 1983. Thesederivatives enable selection of transgenic plant cells on hygromycinonly (pCIB743), or hygromycin and kanamycin (pCIB715, pCIB717).

Methods using either a form of direct gene transfer orAgrobacterium-mediated transfer usually, but not necessarily, areundertaken with a selectable marker which may provide resistance to anantibiotic (e.g., kanamycin, hygromycin or methotrexate) or a herbicide(e.g., phosphinothricin). The choice of selectable marker for planttransformation is not, however, critical to the invention.

For certain plant species, different antibiotic or herbicide selectionmarkers may be preferred. Selection markers used routinely intransformation include the nptII gene which confers resistance tokanamycin and related antibiotics (Messing & Vierra, 1982; Bevan et al.,1983), the bar gene which confers resistance to the herbicidephosphinothricin (White et al., 1990, Spencer et al., 1990), the hphgene which confers resistance to the antibiotic hygromycin (Blochinger &Diggelmann), and the dhfr gene, which confers resistance to methotrexate(Bourouis et al., 1983).

One such vector useful for direct gene transfer techniques incombination with selection by the herbicide Basta (or phosphinothricin)is pCIB3064. This vector is based on the plasmid pCIB246, whichcomprises the CaMV 35S promoter in operational fusion to the E. coli GUSgene and the CaMV 35S transcriptional terminator and is described in thePCT published application WO 93/07278, herein incorporated by reference.One gene useful for conferring resistance to phosphinothricin is the bargene from Streptomyces viridochromogenes (Thompson et al., 1987). Thisvector is suitable for the cloning of plant expression cassettescontaining their own regulatory signals.

An additional transformation vector is pSOG35 which utilizes the E. coligene dihydrofolate reductase (DHFR) as a selectable marker conferringresistance to methotrexate. PCR was used to amplify the 35S promoter(about 800 bp), intron 6 from the maize Adh1 gene (about 550 bp) and 18bp of the GUS untranslated leader sequence from pSOG10. A 250 byfragment encoding the E. coli dihydrofolate reductase type II gene wasalso amplified by PCR and these two PCR fragments were assembled with aSacI-PstI fragment from pBI221 (Clontech) which comprised the pUC19vector backbone and the nopaline synthase terminator. Assembly of thesefragments generated pSOG19 which contains the 35S promoter in fusionwith the intron 6 sequence, the GUS leader, the DHFR gene and thenopaline synthase terminator. Replacement of the GUS leader in pSOG19with the leader sequence from Maize Chlorotic Mottle Virus check (MCMV)generated the vector pSOG35. pSOG19 and pSOG35 carry the pUC-derivedgene for ampicillin resistance and have HindIII, SphI, PstI and EcoRIsites available for the cloning of foreign sequences.

IV. Production and Characterization of Stably Transformed Plants

Transgenic plant cells are then placed in an appropriate selectivemedium for selection of transgenic cells which are then grown to callus.Shoots are grown from callus and plantlets generated from the shoot bygrowing in rooting medium. The various constructs normally will bejoined to a marker for selection in plant cells. Conveniently, themarker may be resistance to a biocide (particularly an antibiotic, suchas kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide,or the like). The particular marker used will allow for selection oftransformed cells as compared to cells lacking the DNA which has beenintroduced. Components of DNA constructs including transcriptioncassettes of this invention may be prepared from sequences which arenative (endogenous) or foreign (exogenous) to the host. By “foreign” itis meant that the sequence is not found in the wild-type host into whichthe construct is introduced. Heterologous constructs will contain atleast one region which is not native to the gene from which thetranscription-initiation-region is derived.

To confirm the presence of the transgenes in transgenic cells andplants, a variety of assays may be performed. Such assays include, forexample, “molecular biological” assays well known to those of skill inthe art, such as Southern and Northern blotting, in situ hybridizationand nucleic acid-based amplification methods such as PCR or RT-PCR;“biochemical” assays, such as detecting the presence of a proteinproduct, e.g., by immunological means (ELISAs and Western blots) or byenzymatic function; plant part assays, such as seed assays; and also, byanalyzing the phenotype of the whole regenerated plant, e.g., fordisease or pest resistance.

DNA may be isolated from cell lines or any plant parts to determine thepresence of the preselected nucleic acid segment through the use oftechniques well known to those skilled in the art. Note that intactsequences will not always be present, presumably due to rearrangement ordeletion of sequences in the cell.

The presence of nucleic acid elements introduced through the methods ofthis invention may be determined by polymerase chain reaction (PCR).Using this technique discreet fragments of nucleic acid are amplifiedand detected by gel electrophoresis. This type of analysis permits oneto determine whether a preselected nucleic acid segment is present in astable transformant, but does not prove integration of the introducedpreselected nucleic acid segment into the host cell genome. In addition,it is not possible using PCR techniques to determine whethertransformants have exogenous genes introduced into different sites inthe genome, i.e., whether transformants are of independent origin. It iscontemplated that using PCR techniques it would be possible to clonefragments of the host genomic DNA adjacent to an introduced preselectedDNA segment.

Positive proof of DNA integration into the host genome and theindependent identities of transformants may be determined using thetechnique of Southern hybridization. Using this technique specific DNAsequences that were introduced into the host genome and flanking hostDNA sequences can be identified. Hence the Southern hybridizationpattern of a given transformant serves as an identifying characteristicof that transformant. In addition it is possible through Southernhybridization to demonstrate the presence of introduced preselected DNAsegments in high molecular weight DNA, i.e., confirm that the introducedpreselected DNA segment has been integrated into the host cell genome.The technique of Southern hybridization provides information that isobtained using PCR, e.g., the presence of a preselected DNA segment, butalso demonstrates integration into the genome and characterizes eachindividual transformant.

It is contemplated that using the techniques of dot or slot blothybridization which are modifications of Southern hybridizationtechniques one could obtain the same information that is derived fromPCR, e.g., the presence of a preselected DNA segment.

Both PCR and Southern hybridization techniques can be used todemonstrate transmission of a preselected DNA segment to progeny. Inmost instances the characteristic Southern hybridization pattern for agiven transformant will segregate in progeny as one or more Mendeliangenes (Spencer et al., 1992); Laursen et al., 1994) indicating stableinheritance of the gene. The nonchimeric nature of the callus and theparental transformants (R₀) was suggested by germline transmission andthe identical Southern blot hybridization patterns and intensities ofthe transforming DNA in callus, R₀ plants and R₁ progeny that segregatedfor the transformed gene.

Whereas DNA analysis techniques may be conducted using DNA isolated fromany part of a plant, RNA may only be expressed in particular cells ortissue types and hence it will be necessary to prepare RNA for analysisfrom these tissues. PCR techniques may also be used for detection andquantitation of RNA produced from introduced preselected DNA segments.In this application of PCR it is first necessary to reverse transcribeRNA into DNA, using enzymes such as reverse transcriptase, and thenthrough the use of conventional PCR techniques amplify the DNA. In mostinstances PCR techniques, while useful, will not demonstrate integrityof the RNA product. Further information about the nature of the RNAproduct may be obtained by Northern blotting. This technique willdemonstrate the presence of an RNA species and give information aboutthe integrity of that RNA. The presence or absence of an RNA species canalso be determined using dot or slot blot Northern hybridizations. Thesetechniques are modifications of Northern blotting and will onlydemonstrate the presence or absence of an RNA species.

While Southern blotting and PCR may be used to detect the preselectedDNA segment in question, they do not provide information as to whetherthe preselected DNA segment is being expressed. Expression may beevaluated by specifically identifying the protein products of theintroduced preselected DNA segments or evaluating the phenotypic changesbrought about by their expression.

Assays for the production and identification of specific proteins maymake use of physical-chemical, structural, functional, or otherproperties of the proteins. Unique physical-chemical or structuralproperties allow the proteins to be separated and identified byelectrophoretic procedures, such as native or denaturing gelelectrophoresis or isoelectric focusing, or by chromatographictechniques such as ion exchange or gel exclusion chromatography. Theunique structures of individual proteins offer opportunities for use ofspecific antibodies to detect their presence in formats such as an ELISAassay. Combinations of approaches may be employed with even greaterspecificity such as Western blotting in which antibodies are used tolocate individual gene products that have been separated byelectrophoretic techniques. Additional techniques may be employed toabsolutely confirm the identity of the product of interest such asevaluation by amino acid sequencing following purification. Althoughthese are among the most commonly employed, other procedures may beadditionally used.

Assay procedures may also be used to identify the expression of proteinsby their functionality, especially the ability of enzymes to catalyzespecific chemical reactions involving specific substrates and products.These reactions may be followed by providing and quantifying the loss ofsubstrates or the generation of products of the reactions by physical orchemical procedures. Examples are as varied as the enzyme to beanalyzed.

Very frequently the expression of a gene product is determined byevaluating the phenotypic results of its expression. These assays alsomay take many forms including but not limited to analyzing changes inthe chemical composition, morphology, or physiological properties of theplant. Morphological changes may include greater stature or thickerstalks. Most often changes in response of plants or plant parts toimposed treatments are evaluated under carefully controlled conditionstermed bioassays.

V. Uses of Transgenic Plants

Once an expression cassette of the invention has been transformed into aparticular plant species, it may be propagated in that species or movedinto other varieties of the same species, particularly includingcommercial varieties, using traditional breeding techniques.Particularly preferred plants of the invention include the agronomicallyimportant crops listed above. The genetic properties engineered into thetransgenic seeds and plants described above are passed on by sexualreproduction and can thus be maintained and propagated in progenyplants. The present invention also relates to a transgenic plant cell,tissue, organ, seed or plant part obtained from the transgenic plant.Also included within the invention are transgenic descendants of theplant as well as transgenic plant cells, tissues, organs, seeds andplant parts obtained from the descendants.

Preferably, the expression cassette in the transgenic plant is sexuallytransmitted. In one preferred embodiment, the coding sequence issexually transmitted through a complete normal sexual cycle of the R0plant to the R1 generation. Additionally preferred, the expressioncassette is expressed in the cells, tissues, seeds or plant of atransgenic plant in an amount that is different than the amount in thecells, tissues, seeds or plant of a plant which only differs in that theexpression cassette is absent.

The transgenic plants produced herein are thus expected to be useful fora variety of commercial and research purposes. Transgenic plants can becreated for use in traditional agriculture to possess traits beneficialto the grower (e.g., agronomic traits such as resistance to waterdeficit, pest resistance, herbicide resistance or increased yield),beneficial to the consumer of the grain harvested from the plant (e.g.,improved nutritive content in human food or animal feed; increasedvitamin, amino acid, and antioxidant content; the production ofantibodies (passive immunization) and nutriceuticals), or beneficial tothe food processor (e.g., improved processing traits). In such uses, theplants are generally grown for the use of their grain in human or animalfoods. Additionally, the use of root-specific promoters in transgenicplants can provide beneficial traits that are localized in theconsumable (by animals and humans) roots of plants such as carrots,parsnips, and beets. However, other parts of the plants, includingstalks, husks, vegetative parts, and the like, may also have utility,including use as part of animal silage or for ornamental purposes.Often, chemical constituents (e.g., oils or starches) of maize and othercrops are extracted for foods or industrial use and transgenic plantsmay be created which have enhanced or modified levels of suchcomponents.

Transgenic plants may also find use in the commercial manufacture ofproteins or other molecules, where the molecule of interest is extractedor purified from plant parts, seeds, and the like. Cells or tissue fromthe plants may also be cultured, grown in vitro, or fermented tomanufacture such molecules.

The transgenic plants may also be used in commercial breeding programs,or may be crossed or bred to plants of related crop species.Improvements encoded by the expression cassette may be transferred,e.g., from maize cells to cells of other species, e.g., by protoplastfusion.

The transgenic plants may have many uses in research or breeding,including creation of new mutant plants through insertional mutagenesis,in order to identify beneficial mutants that might later be created bytraditional mutation and selection. An example would be the introductionof a recombinant DNA sequence encoding a transposable element that maybe used for generating genetic variation. The methods of the inventionmay also be used to create plants having unique “signature sequences” orother marker sequences which can be used to identify proprietary linesor varieties.

Thus, the transgenic plants and seeds according to the invention can beused in plant breeding which aims at the development of plants withimproved properties conferred by the expression cassette, such astolerance of drought, disease, or other stresses. The various breedingsteps are characterized by well-defined human intervention such asselecting the lines to be crossed, directing pollination of the parentallines, or selecting appropriate descendant plants. Depending on thedesired properties different breeding measures are taken. The relevanttechniques are well known in the art and include but are not limited tohybridization, inbreeding, backcross breeding, ultilane breeding,variety blend, interspecific hybridization, aneuploid techniques, etc.Hybridization techniques also include the sterilization of plants toyield male or female sterile plants by mechanical, chemical orbiochemical means. Cross pollination of a male sterile plant with pollenof a different line assures that the genome of the male sterile butfemale fertile plant will uniformly obtain properties of both parentallines. Thus, the transgenic seeds and plants according to the inventioncan be used for the breeding of improved plant lines which for exampleincrease the effectiveness of conventional methods such as herbicide orpesticide treatment or allow to dispense with said methods due to theirmodified genetic properties. Alternatively new crops with improvedstress tolerance can be obtained which, due to their optimized genetic“equipment”, yield harvested product of better quality than productswhich were not able to tolerate comparable adverse developmentalconditions.

Polynucleotides derived from nucleotide sequences of the presentinvention having any of the nucleotide sequences of SEQ ID NOs: 1 to SEQID NO: 1597, 5927, 5940, 5941, 5945-5958 are useful to detect thepresence in a test sample of at least one copy of a nucleotide sequencecontaining the same or substantially the same sequence, or a fragment,complement, or variant thereof. The sequence of the probes and/orprimers of the instant invention need not be identical to those providedin the Sequence Listing or the complements thereof. Some variation inprobe or primer sequence and/or length can allow additional familymembers to be detected, as well as orthologous genes and moretaxonomically distant related sequences. Similarly probes and/or primersof the invention can include additional nucleotides that serve as alabel for detecting duplexes, for isolation of duplexed polynucleotides,or for cloning purposes.

Preferred probes and primers of the invention include isolated,purified, or recombinant polynucleotides containing a contiguous span ofbetween at least 12 to at least 1000 nucleotides of any nucleotidsequence which is substantially similar, and preferably has at leastbetween 70% and 99% sequence identity to any one of SEQ ID NOs: 1 to1597, 5927, 5940, 5941, 5945-5958 and further of any nucleotide sequencewhich is substantially similar, and preferably has at least between 70%and 99% sequence identity to any one of SEQ ID NO: 1598 to 2672, 5959,5972, 5973, 5977-5990 and 6001 representing promoter sequences, or thecomplements thereof, with each individual number of nucleotides withinthis range also being part of the invention. Preferred are isolated,purified, or recombinant polynucleotides containing a contiguous span ofat least 12, 15, 18, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 150,200, 300, 400, 500, 750, or 1000 nucleotides of any nucleotide sequencewhich is substantially similar, and preferably has at least between 70%and 99% sequence identity to any one of SEQ ID NOs: 1 to 1597, 5927,5940, 5941, 5945-5958 and further of any nucleotide sequence which issubstantially similar, and preferably has at least between 70% and 99%sequence identity to any one of SEQ ID NO: 1598 to 2672, 5959, 5972,5973, 5977-5990 and 6001 representing promoter sequences, or thecomplements thereof. The appropriate length for primers and probes willvary depending on the application. For use as PCR primers, probes are12-40 nucleotides, preferably 18-30 nucleotides long. For use inmapping, probes are 50 to 500 nucleotides, preferably 100-250nucleotides long. For use in Southern hybridizations, probes as long asseveral kilobases can be used. The appropriate length for primers andprobes under a particular set of assay conditions may be empiricallydetermined by one of skill in the art.

The primers and probes can be prepared by any suitable method,including, for example, cloning and restriction of appropriate sequencesand direct chemical synthesis by a method such as the phosphodiestermethod of Narang et al. (Meth Enzymol 68: 90 (1979)), thediethylphosphoramidite method, the triester method of Matteucci et al.(J Am Chem Soc 103: 3185 (1981)), or according to Urdea et al. (ProcNatl Acad 80: 7461 (1981)), the solid support method described in EP 0707 592, or using commercially available automated oligonucleotidesynthesizers.

Detection probes are generally nucleotide sequences or unchargednucleotide analogs such as, for example peptide nucleotides which aredisclosed in International Patent Application WO 92/20702, morpholinoanalogs which are described in U.S. Pat. Nos. 5,185,444, 5,034,506 and5,142,047. The probe may have to be rendered “non-extendable” such thatadditional dNTPs cannot be added to the probe. Analogs are usuallynon-extendable, and nucleotide probes can be rendered non-extendable bymodifying the 3′ end of the probe such that the hydroxyl group is nolonger capable of participating in elongation. For example, the 3′ endof the probe can be functionalized with the capture or detection labelto thereby consume or otherwise block the hydroxyl group. Alternatively,the 3′ hydroxyl group simply can be cleaved, replaced or modified so asto render the probe non-extendable.

Any of the polynucleotides of the present invention can be labeled, ifdesired, by incorporating a label detectable by spectroscopic,photochemical, biochemical, immunochemical, or chemical means. Forexample, useful labels include radioactive substances (³²P, ³⁵S, ³H,¹²⁵I) fluorescent dyes (5-bromodesoxyuridine, fluorescein,acetylaminofluorene, digoxigenin) or biotin. Preferably, polynucleotidesare labeled at their 3′ and 5′ ends. Examples of non-radioactivelabeling of nucleotide fragments are described in the French patent No.FR-7810975 and by Urdea et al. (Nuc Acids Res 16:4937 (1988)). Inaddition, the probes according to the present invention may havestructural characteristics such that they allow the signalamplification, such structural characteristics being, for example,branched DNA probes as described in EP 0 225 807.

A label can also be used to capture the primer so as to facilitate theimmobilization of either the primer or a primer extension product, suchas amplified DNA, on a solid support. A capture label is attached to theprimers or probes and can be a specific binding member that forms abinding pair with the solid's phase reagent's specific binding member,for example biotin and streptavidin. Therefore depending upon the typeof label carried by a polynucleotide or a probe, it may be employed tocapture or to detect the target DNA. Further, it will be understood thatthe polynucleotides, primers or probes provided herein, may, themselves,serve as the capture label. For example, in the case where a solid phasereagent's binding member is a nucleotide sequence, it may be selectedsuch that it binds a complementary portion of a primer or probe tothereby immobilize the primer or probe to the solid phase. In caseswhere a polynucleotide probe itself serves as the binding member, thoseskilled in the art will recognize that the probe will contain a sequenceor “tail” that is not complementary to the target. In the case where apolynucleotide primer itself serves as the capture label, at least aportion of the primer will be free to hybridize with a nucleotide on asolid phase. DNA labeling techniques are well known in the art.

Any of the polynucleotides, primers and probes of the present inventioncan be conveniently immobilized on a solid support. Solid supports areknown to those skilled in the art and include the walls of wells of areaction tray, test tubes, polystyrene beads, magnetic beads,nitrocellulose strips, membranes, microparticles such as latexparticles, sheep (or other animal) red blood cells, duracytes andothers. The solid support is not critical and can be selected by oneskilled in the art. Thus, latex particles, microparticles, magnetic ornon-magnetic beads, membranes, plastic tubes, walls of microtiter wells,glass or silicon chips, sheep (or other suitable animal's) red bloodcells and duracytes are all suitable examples. Suitable methods forimmobilizing nucleotides on solid phases include ionic, hydrophobic,covalent interactions and the like. A solid support, as used herein,refers to any material that is insoluble, or can be made insoluble by asubsequent reaction. The solid support can be chosen for its intrinsicability to attract and immobilize the capture reagent. Alternatively,the solid phase can retain an additional receptor that has the abilityto attract and immobilize the capture reagent. The additional receptorcan include a charged substance that is oppositely charged with respectto the capture reagent itself or to a charged substance conjugated tothe capture reagent. As yet another alternative, the receptor moleculecan be any specific binding member which is immobilized upon (attachedto) the solid support and which has the ability to immobilize thecapture reagent through a specific binding reaction. The receptormolecule enables the indirect binding of the capture reagent to a solidsupport material before the performance of the assay or during theperformance of the assay. The solid phase thus can be a plastic,derivatized plastic, magnetic or non-magnetic metal, glass or siliconsurface of a test tube, microtiter well, sheet, bead, microparticle,chip, sheep (or other suitable animal's) red blood cells, duracytes andother configurations known to those of ordinary skill in the art. Thepolynucleotides of the invention can be attached to or immobilized on asolid support individually or in groups of at least 2, 5, 8, 10, 12, 15,20, or 25 distinct polynucleotides of the invention to a single solidsupport. In addition, polynucleotides other than those of the inventionmay be attached to the same solid support as one or more polynucleotidesof the invention.

The polynucleotides of the invention that are expressed or repressed inresponse to environmental stimuli such as, for example, stress ortreatment with chemicals or pathogens or at different developmentalstages can be identified by employing an array of nucleic acid samples,e.g., each sample having a plurality of oligonucleotides, and eachplurality corresponding to a different plant gene, on a solid substrate,e.g., a DNA chip, and probes corresponding to nucleic acid expressed in,for example, one or more plant tissues and/or at one or moredevelopmental stages, e.g., probes corresponding to nucleic acidexpressed in seed of a plant relative to control nucleic acid fromsources other than seed. Thus, genes that are upregulated ordownregulated in the majority of tissues at a majority of developmentalstages, or upregulated or downregulated in one tissue such as in seed,can be systematically identified. The probes may also correspond tonucleic acid expressed in respone to a defined treatment such as, forexample, a treatment with a variety of plant hormones or the exposure tospecific environmental conditions involving, for example, an abioticstress or exposure to light.

Specifically, labeled rice cRNA probes were hybridized to the rice DNAarray, expression levels were determined by laser scanning and then ricegenes were identified that had a particular expression pattern. The riceoligonucleotide probe array consists of probes from over 18,000 uniquerice genes, which covers approximately 40-50% of the genome. This genomearray permits a broader, more complete and less biased analysis of geneexpression.

Consequently, the invention also deals with a method for detecting thepresence of a polynucleotide including a nucleotide sequence which issubstantially similar to a nucleotide sequence given in SEQ ID NOs: 1 toSEQ ID NO: 6001, or a fragment or a variant thereof, or a complementarysequence thereto, in a sample, the method including the following stepsof:

-   -   (a) bringing into contact a nucleotide probe or a plurality of        nucleotide probes which can hybridize with a polynucleotide        having a nucleotide sequence which is substantially similar to a        nucleotide sequence given in SEQ ID NOs: 1 to SEQ ID NO: 6001, a        fragment or a variant thereof, or a complementary sequence        thereto and the sample to be assayed.    -   (b) detecting the hybrid complex formed between the probe and a        nucleotide in the sample.

The invention further concerns a kit for detecting the presence of apolynucleotide including a nucleotide sequence which is substantiallysimilar to a nucleotide sequence given in SEQ ID NOs: 1 to SEQ ID NO:6001, a fragment or a variant thereof, or a complementary sequencethereto, in a sample, the kit including a nucleotide probe or aplurality of nucleotide probes which can hybridize with a nucleotidesequence included in a polynucleotide, which nucleotide sequence issubstantially similar to a nucleotide sequence given in of SEQ ID NOs: 1to SEQ ID NO: 6001, a fragment or a variant thereof, or a complementarysequence thereto and, optionally, the reagents necessary for performingthe hybridization reaction.

In a first preferred embodiment of this detection method and kit, thenucleotide probe or the plurality of nucleotide probes are labeled witha detectable molecule. In a second preferred embodiment of the methodand kit, the nucleotide probe or the plurality of nucleotide probes hasbeen immobilized on a substrate.

The isolated polynucleotides of the invention can be used to createvarious types of genetic and physical maps of the genome of rice orother plants. Such maps are used to devise positional cloning strategiesfor isolating novel genes from the mapped crop species. The sequences ofthe present invention are also useful for chromosome mapping, chromosomeidentification, tagging of genes which are tissue-specificallyexpressed.

The isolated polynucleotides of the invention can further be used asprobes for identifying polymorphisms associated with phenotypes ofinterest. Briefly, total DNA is isolated from an individual or isogenicline, cleaved with one or more restriction enzymes, separated accordingto mass, transferred to a solid support, and hybridized with a probemolecule according to the invention. The pattern of fragmentshybridizing to a probe molecule is compared for DNA from differentindividuals or lines, where differences in fragment size signals apolymorphism associated with a particular nucleotide sequence accordingto the present invention. After identification of polymorphic sequences,linkage studies can be conducted. After identification of manypolymorphisms using a nucleotide sequence according to the invention,linkage studies can be conducted by using the individuals showingpolymorphisms as parents in crossing programs. Recombinants, F₂ progenyrecombinants or recombinant inbreds, can then be analyzed using the samerestriction enzyme/hybridization procedure. The order of DNApolymorphisms along the chromosomes can be inferred based on thefrequency with which they are inherited together versus inheritedindependently. The closer together two polymorphisms occur in achromosome, the higher the probability that they are inherited together.Integration of the relative positions of polymorphisms and associatedmarker sequences produces a genetic map of the species, where thedistances between markers reflect the recombination frequencies in thatchromosome segment. Preferably, the polymorphisms and marker sequencesare sufficiently numerous to produce a genetic map of sufficiently highresolution to locate one or more loci of interest.

The use of recombinant inbred lines for such genetic mapping isdescribed for rice (Oh et al., Mol Cells 8:175 (1998); Nandi et al., MolGen Genet 255:1 (1997); Wang et al., Genetics 136:1421 (1994)), sorghum(Subudhi et al., Genome 43:240 (2000)), maize (Burr et al., Genetics118:519 (1998); Gardiner et al., Genetics 134:917 (1993)), andArabidopsis (Methods in Molecular Biology, Martinez-Zapater and Salinas,eds., 82:137-146, (1998)). However, this procedure is not limited toplants and can be used for other organisms such as yeast or other fungi,or for oomycetes or other protistans.

The nucleotide sequences of the present invention can also be used forsimple sequence repeat identification, also known as single sequencerepeat, (SSR) mapping. SSR mapping in rice has been described by Miyaoet al. (DNA Res 3:233 (1996)) and Yang et al. (Mol Gen Genet 245:187(1994)), and in maize by Ahn et al. (Mol Gen Genet 241:483 (1993)). SSRmapping can be achieved using various methods. In one instance,polymorphisms are identified when sequence specific probes flanking anSSR contained within an sequence of the invention are made and used inpolymerase chain reaction (PCR) assays with template DNA from two ormore individuals or, in plants, near isogenic lines. A change in thenumber of tandem repeats between the SSR-flanking sequence producesdifferently sized fragments (U.S. Pat. No. 5,766,847). Alternatively,polymorphisms can be identified by using the PCR fragment produced fromthe SSR-flanking sequence specific primer reaction as a probe againstSouthern blots representing different individuals (Refseth et al.,Electrophoresis 18:1519 (1997)). Rice SSRs were used to map a molecularmarker closely linked to a nuclear restorer gene for fertility in riceas described by Akagi et al. (Genome 39:205 (1996)).

The nucleotide sequences of the present invention can be used toidentify and develop a variety of microsatellite markers, including theSSRs described above, as genetic markers for comparative analysis andmapping of genomes. The nucleotide sequences of the present inventioncan be used in a variation of the SSR technique known as inter-SSR(ISSR), which uses microsatellite oligonucleotides as primers to amplifygenomic segments different from the repeat region itself (Zietkiewicz etal., Genomics 20:176 (1994)). ISSR employs oligonucleotides based on asimple sequence repeat anchored or not at their 5′- or 3′-end by two tofour arbitrarily chosen nucleotides, which triggers site-specificannealing and initiates PCR amplification of genomic segments which areflanked by inversely orientated and closely spaced repeat sequences. Inone embodiment of the present invention, microsatellite markers derivedfrom the nucleotide sequences disclosed in the Sequence Listing, orsubstantially similar sequences or allelic variants thereof, may be usedto detect the appearance or disappearance of markers indicating genomicinstability as described by Leroy et al. (Electron. J Biotechnol, 3(2),(2000), available at the website of the Electronic Journal ofBiotechnology), where alteration of a fingerprinting pattern indicatedloss of a marker corresponding to a part of a gene involved in theregulation of cell proliferation. Microsatellite markers derived fromnucleotide sequences as provided in the Sequence Listing will be usefulfor detecting genomic alterations such as the change observed by Leroyet al. (Electron. J Biotechnol, 3(2), supra (2000)) which appeared to bethe consequence of microsatellite instability at the primer binding siteor modification of the region between the microsatellites, andillustrated somaclonal variation leading to genomic instability.Consequently, the nucleotide sequences of the present invention areuseful for detecting genomic alterations involved in somaclonalvariation, which is an important source of new phenotypes.

In addition, because the genomes of closely related species are largelysyntenic (that is, they display the same ordering of genes within thegenome), these maps can be used to isolate novel alleles from wildrelatives of crop species by positional cloning strategies. This sharedsynteny is very powerful for using genetic maps from one species to mapgenes in another. For example, a gene mapped in rice providesinformation for the gene location in maize and wheat.

The various types of maps discussed above can be used with thenucleotide sequences of the invention to identify Quantitative TraitLoci (QTLs) for a variety of uses, including marker-assisted breeding.Many important crop traits are quantitative traits and result from thecombined interactions of several genes. These genes reside at differentloci in the genome, often on different chromosomes, and generallyexhibit multiple alleles at each locus. Developing markers, tools, andmethods to identify and isolate the QTLs enables marker-assistedbreeding to enhance traits of interest or suppress undesirable traitsthat interfere with a desired effect. The nucleotide sequences asprovided in the Sequence Listing can be used to generate markers,including single-sequence repeats (SSRs) and microsatellite markers forQTLs of interest to assist marker-assisted breeding. The nucleotidesequences of the invention can be used to identify QTLs and isolatealleles as described by Li et al. in a study of QTLs involved inresistance to a pathogen of rice. (Li et al., Mol Gen Genet 261:58(1999)). In addition to isolating QTL alleles in rice, other cereals,and other monocot and dicot crop species, the nucleotide sequences ofthe invention can also be used to isolate alleles from the correspondingQTL(s) of wild relatives. Transgenic plants having various combinationsof QTL alleles can then be created and the effects of the combinationsmeasured. Once an ideal allele combination has been identified, cropimprovement can be accomplished either through biotechnological means orby directed conventional breeding programs. (Flowers et al., J Exp Bot51:99 (2000); Tanksley and McCouch, Science 277:1063 (1997)).

In another embodiment the nucleotide sequences of the invention can beused to help create physical maps of the genome of maize, Arabidopsisand related species. Where the nucleotide sequences of the inventionhave been ordered on a genetic map, as described above, then thenucleotide sequences of the invention can be used as probes to discoverwhich clones in large libraries of plant DNA fragments in YACs, PACs,etc. contain the same nucleotide sequences of the invention or similarsequences, thereby facilitating the assignment of the large DNAfragments to chromosomal positions. Subsequently, the large BACs, YACs,etc. can be ordered unambiguously by more detailed studies of theirsequence composition and by using their end or other sequence to findthe identical sequences in other cloned DNA fragments (Mozo et al., NatGenet 22:271 (1999)). Overlapping DNA sequences in this way allowsassembly of large sequence contigs that, when sufficiently extended,provide a complete physical map of a chromosome. The nucleotidesequences of the invention themselves may provide the means of joiningcloned sequences into a contig, and are useful for constructing physicalmaps.

In another embodiment, the nucleotide sequences of the present inventionmay be useful in mapping and characterizing the genomes of othercereals. Rice has been proposed as a model for cereal genome analysis(Havukkala, Curr Opin Genet Devel 6:711 (1996)), based largely on itssmaller genome size and higher gene density, combined with theconsiderable conserved gene order among cereal genomes (Ahn et al., MolGen Genet 241:483 (1993)). The cereals demonstrate both generalconservation of gene order (synteny) and considerable sequence homologyamong various cereal gene families. This suggests that studies on thefunctions of genes or proteins from rice that are tissue-specificallyexpressed could lead to the identification of orthologous genes orproteins in other cereals, including maize, wheat, secale, sorghum,barley, millet, teff, milo, triticale, flax, gramma grass, Tripsacumsp., and teosinte. The nucleotide sequences according to the inventioncan also be used to physically characterize homologous chromosomes inother cereals, as described by Sarma et al. (Genome 43:191 (2000)), andtheir use can be extended to non-cereal monocots such as sugarcane,grasses, and lilies.

Given the synteny between rice and other cereal genomes, the nucleotidesequences of the present invention can be used to obtain molecularmarkers for mapping and, potentially, for positional cloning. Kilian etal. described the use of probes from the rice genomic region of interestto isolate a saturating number of polymorphic markers in barley, whichwere shown to map to syntenic regions in rice and barley, suggestingthat the nucleotide sequences of the invention derived from the ricegenome would be useful in positional cloning of syntenic genes ofinterest from other cereal species. (Kilian, et al., Nucl Acids Res23:2729 (1995); Kilian, et al., Plant Mol Biol 35:187 (1997)). Syntenybetween rice and barley has recently been reported in the area of thecarrying malting quality QTLs (Han, et al., Genome 41:373 (1998)), anduse of synteny between cereals for positional cloning efforts is likelyto add considerable value to rice genome analysis. Likewise, mapping ofthe ligules region of sorghum was facilitated using molecular markersfrom a syntenic region of the rice genome. (Zwick, et al., Genetics148:1983 (1998)).

Rice marker technology utilizing the nucleotide sequences of the presentinvention can also be used to identify QTL alleles for a trait ofinterest from a wild relative of cultivated rice, for example asdescribed by Xiao, et al. (Genetics 150:899 (1998)). Wild relatives ofdomesticated plants represent untapped pools of genetic resources forabiotic and biotic stress resistance, apomixis and other breedingstrategies, plant architecture, determinants of yield, secondarymetabolites, and other valuable traits. In rice, Xiao et al. (supra)used molecular markers to introduce an average of approximately 5% ofthe genome of a wild relative, and the resulting plants were scored forphenotypes such as plant height, panicle length and 1000-grain weight.Trait-improving alleles were found for all phenotypes except plantheight, where any change is considered negative. Of the 35trait-improving alleles, Xiao et al. found that 19 had no effect onother phenotypes whereas 16 had deleterious effects on other traits. Thenucleotide sequences of the invention such as those provided in theSequence Listing can be employed as molecular markers to identify QTLalleles for trait of interest from a wild relative, by which thesevaluable traits can be introgressed from wild relatives using methodsincluding, but not limited to, that described by Xiao et al. ((1998)supra). Accordingly, the nucleotide sequences of the invention can beemployed in a variety of molecular marker technologies for yieldimprovement.

Following the procedures described above to identify polymorphisms, andusing a plurality of the nucleotide sequences of the invention, anyindividual (or line) can be genotyped. Genotyping a large number of DNApolymorphisms such as single nucleotide polymorphisms (SNPs), inbreeding lines makes it possible to find associations between certainpolymorphisms or groups of polymorphisms, and certain phenotypes. Inaddition to sequence polymorphisms, length polymorphisms such as tripletrepeats are studied to find associations between polymorphism andphenotype. Genotypes can be used for the identification of particularcultivars, varieties, lines, ecotypes, and genetically modified plantsor can serve as tools for subsequent genetic studies of complex traitsinvolving multiple phenotypes.

The patent publication WO95/35505 and U.S. Pat. Nos. 5,445,943 and5,410,270 describe scanning multiple alleles of a plurality of lociusing hybridization to arrays of oligonucleotides. The nucleotidesequences of the invention are suitable for use in genotyping techniquesuseful for each of the types of mapping discussed above.

In a preferred embodiment, the nucleotide sequences of the invention areuseful for identifying and isolating a least one unique stretch ofprotein-encoding nucleotide sequence. The nucleotide sequences of theinvention are compared with other coding sequences having sequencesimilarity with the sequences provided in the Sequence Listing, using aprogram such as BLAST. Comparison of the nucleotide sequences of theinvention with other similar coding sequences permits the identificationof one or more unique stretches of coding sequences encoding proteinsthat are tissue-specifically expressed and that are not identical to thecorresponding coding sequence being screened. Preferably, a uniquestretch of coding sequence of about 25 base pairs (bp) long isidentified, more preferably 25 bp, or even more preferably 22 bp, or 20bp, or yet even more preferably 18 bp or 16 bp or 14 bp. In oneembodiment, a plurality of nucleotide sequences is screened to identifyunique coding sequences accroding to the invention. In one embodiment,one or more unique coding sequences accroding to the invention can beapplied to a chip as part of an array, or used in a non-chip arraysystem. In a further embodiment, a plurality of unique coding sequencesaccroding to the invention is used in a screening array. In anotherembodiment, one or more unique coding sequences accroding to theinvention can be used as immobilized or as probes in solution. In yetanother embodiment, one or more unique coding sequences accroding to theinvention can be used as primers for PCR. In a further embodiment, oneor more unique coding sequences accroding to the invention can be usedas organism-specific primers for PCR in a solution containing DNA from aplurality of sources.

In another embodiment unique stretches of nucleotide sequences accordingto the invention are identified that are preferably about 30 bp, morepreferably 50 bp or 75 bp, yet more preferably 100 bp, 150 bp, 200 bp,250, 500 bp, 750 bp, or 1000 bp. The length of an unique coding sequencemay be chosen by one of skill in the art depending on its intended useand on the characteristics of the nucleotide sequence being used. In oneembodiment, unique coding sequences accroding to the invention may beused as probes to screen libraries to find homologs, orthologs, orparalogs. In another embodiment, unique coding sequences accroding tothe invention may be used as probes to screen genomic DNA or cDNA tofind homologs, orthologs, or paralogs. In yet another embodiment, uniquecoding sequences accroding to the invention may be used to study geneevolution and genome evolution.

The invention also provides a computer readable medium having storedthereon a data structure containing nucleic acid sequences having atleast 70% sequence identity to a nucleic acid sequence selected fromthose listed in SEQ ID Nos: 1-6001, as well as complementary, ortholog,and variant sequences thereof. Storage and use of nucleic acid sequenceson a computer readable medium is well known in the art. See for exampleU.S. Pat. Nos. 6,023,659; 5,867,402; 5,795,716. Examples of such mediuminclude, but are not limited to, magnetic tape, optical disk, CD-ROM,random access memory, volatile memory, non-volatile memory and bubblememory. Accordingly, the nucleic acid sequences contained on thecomputer readable medium may be compared through use of a module thatreceives the sequence information and compares it to other sequenceinformation. Examples of other sequences to which the nucleic acidsequences of the invention may be compared include those maintained bythe National Center for Biotechnology Information (NCBI), accessiblethrough the World Wide Web, and the Swiss Protein Data Bank. A computeris an example of such a module that can read and compare nucleic acidsequence information. Accordingly, the invention also provides themethod of comparing a nucleic acid sequence of the invention to anothersequence. For example, a sequence of the invention may be submitted tothe NCBI for a Blast search as described herein where the sequence iscompared to sequence information contained within the NCBI database anda comparison is returned. The invention also provides nucleic acidsequence information in a computer readable medium that allows theencoded polypeptide to be optimized for a desired property. Examples ofsuch properties include, but are not limited to, increased or decreased:thermal stability, chemical stability, hydrophylicity, hydrophobicity,and the like. Methods for the use of computers to model polypeptides andpolynucleotides having altered activities are well known in the art andhave been reviewed. (Lesyng et al., 1993; Surles et al., 1994; Koehl etal., 1996; Rossi et al., 2001).

EXAMPLE 1 GENECHIP® Standard Protocol 1.1 Quantitation of Total RNA

Total RNA from plant tissue is extracted and quantified.

30 Quantify total RNA using GeneQuant

-   -   1OD₂₆₀=40 mg RNA/ml; A₂₆₀/A₂₈₀=1.9 to about 2.1

2. Run gel to check the integrity and purity of the extracted RNA

1.2 Synthesis of Double-Stranded cDNA

Gibco/BRL SuperScript Choice System for cDNA Synthesis (Cat#1B090-019)was employed to prepare cDNAs. T7-(dT)₂₄ oligonucleotides were preparedand purified by HPLC.(5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)₂₄-3′; SEQ ID NO:4709).

1.2.1 Step 1. Primer Hybridization:

-   -   Incubate at 70° C. for 10 minutes    -   Quick spin and put on ice briefly

1.2.2 Step 2. Temperature Adjustment:

-   -   Incubate at 42° C. for 2 minutes

1.2.3 Step 3. First Strand Synthesis:

-   -   DEPC-water-1:1    -   RNA (10:g final)-10:1    -   T7=(dT)₂₄ Primer (100 pmol final)-1:1 pmol    -   5× 1^(st) strand cDNA buffer-4:1    -   0.1M DTT (10 mM final)-2:1    -   10 mM dNTP mix (500:M final)-1:1    -   Superscript II RT 200 U/:1-1:1    -   Total of 20:1

Mix well

Incubate at 42° C. for 1 hour

1.2.4 Step 4. Second strand synthesis:

-   -   Place reactions on ice, quick spin    -   DEPC-water-91:1    -   5× 2^(nd) strand cDNA buffer-30:1    -   10 mM dNTP mix (250 mM final)-3:1    -   E. coli DNA ligase (10 U/:1)-1:1    -   E. coli DNA polymerase 1-10 U/:1-4:1    -   RnaseH 2U/:1-1:1    -   T4 DNA polymerase 5 U/:1-2:1    -   0.5 M EDTA (0.5 M final)-10:1    -   Total 162:1    -   Mix/spin down/incubate 16° C. for 2 hours

1.2.5 Step 5. Completing the Reaction:

-   -   Incubate at 16° C. for 5 minutes        1.3 Purification of Double Stranded cDNA    -   1. Centrifuge PLG (Phase Lock Gel, EPPENDORF® 5 Prime Inc.,        pI-188233) at 14,000×, transfer 162:1 of cDNA to PLG    -   2. Add 162:1 of Phenol:Chloroform:Isoamyl alcohol (pH 8.0),        centrifuge 2 minutes    -   3. Transfer the supernatant to a fresh 1.5 ml tube, add

Glycogen (5 mg/ml)  2 μl 0.5M NH₄OAC (0.75 × Vol) 120 μl ETOH (2.5 ×Vol, −20° C.) 400 μl

-   -   4. Mix well and centrifuge at 14,000× for 20 minutes    -   5. Remove supernatant, add 0.5 ml 80% EtOH (−20° C.)    -   6. Centrifuge for 5 minutes, air dry or by speed vac for 5-10        minutes    -   7. Add 44:1 DEPC H₂O        Analyze of quantity and size distribution of cDNA        Run a gel using 1:1 of the double-stranded synthesis product        1.4 Synthesis of Biotinylated cRNA    -   (use Enzo BioArray High Yield RNA Transcript Labeling Kit        Cat#900182)

Purified cDNA 22:1 10X Hy buffer  4:1 10X biotin ribonucleotides  4:110X DTT  4:1 10X Rnase inhibitor mix  4:1 20X T7 RNA polymerase  2:1Total 40:1

-   Centrifuge 5 seconds, and incubate for 4 hours at 37° C.-   Gently mix every 30-45 minutes

1.5 Purification and quantification of cRNA

-   (use Qiagen RNEASY® Mini kit Cat #74103)

cRNA  40:1 DEPC H₂O  60:1 RLT buffer 350:1 mix by vortexing EtOH 250:1mix by pipetting Total 700:1 Wait 1 minute or more for the RNA to stickCentrifuge at 2000 rpm for 5 minutes RPE buffer 500:1 Centrifuge at10,000 rpm for 1 minute RPE buffer 500:1 Centrifuge at 10,000 rpm for 1minute Centrifuge at 10,000 rpm for 1 minute to dry the column DEPC H₂O 30:1 Wait for 1 minute, then elute cRNA from by centrifugation, 10K 1minute DEPC H₂O  30:1 Repeat previous step Determine concentration anddilute to 1:g/:1 concentration1.6 Fragmentation of cRNA

cRNA (1:g/:1) 15:1 5X Fragmentation Buffer*  6:1 DEPC H₂O  9:1 30:1 *5xFragmentation Buffer

1M Tris (pH8.1) 4.0 ml MgOAc 0.64 g KOAC 0.98 g DEPC H₂O Total 20 ml

Filter Sterilize

1.7 Array Wash and Staining

-   Stringent Wash Buffer**-   Non-Stringent Wash Buffer***-   SAPE Stain****-   Antibody Stain*****-   Wash on fluidics station using the appropriate antibody    amplification protocol    -   **Stringent Buffer: 12× MES 83.3 ml, 5 M NaCl 5.2 ml, 10% Tween        1.0 ml, H₂O 910 ml, Filter Sterilize    -   ***Non-Stringent Buffer: 20× SSPE 300 ml, 10% Tween 1.0 ml, H₂O        698 ml, Filter Sterilize, Antifoam 1.0.    -   ****SAPE stain: 2× Stain Buffer 600:1, BSA 48:1, SAPE 12:1, H₂O        540:1.    -   *****Antibody Stain: 2× Stain Buffer 300:1, H₂O 266.4:1, BSA        24:1, Goat IgG 6:1, Biotinylated Ab 3.6:1

EXAMPLE 2 Characterization of Gene Expression Profiles During OryzaPlant Development

A rice gene array (proprietary to Affymetrix) and probes derived fromrice RNA extracted from different tissues and developmental stages wereused to identify the expression profile of genes on the chip. The ricearray contains over 23,000 genes (approximately 18,000 unique genes) orroughly 50% of the rice genome and is similar to the ArabidopsisGENECHIP® (Affymetrix) with the exception that the 16 oligonucleotideprobe sets do not contain mismatch probe sets. The level of expressionis therefore determined by internal software that analyzes the intensitylevel of the 16 probe sets for each gene. The highest and lowest probesare removed if they do not fit into a set of predefined statisticalcriteria and the remaining sets are averaged to give an expressionvalue. The final expression values are normalized by software, asdescribed below. The advantages of a gene chip in such an analysisinclude a global gene expression analysis, quantitative results, ahighly reproducible system, and a higher sensitivity than Northern blotanalyses.

Total RNA was isolated from 29 samples at different developmental stages(see below).

germinating seed root germinating seed leaf 3-4 leaf arial roottillering leaf tillering arial tillering panicle 1-3 panicle 4-7 panicle8-14 panicle 15-20 Panicle panicle emergence leaf booting arial bootingroot booting root panicle emergence stem panicle emergence Inflorescencestem mature root mature leaf mature stem senescence leaf senescenceEmbryo Endosperm seed coat Aleurone seed milk seed soft seed hard

EXAMPLE 2.1 Preparation of RNA

Total RNA is prepared from the frozen samples using Qiagen RNEASY®columns (Valencia, Calif.) and precipitated overnight at −20 ° C. afterthe addition of 0.25 volumes of 10M LiCl₂. Pellets are washed with 70%EtOH, air dried and resuspended in RNase-free water. Alternatively,total RNA is prepared using the “Pine Tree method” (Chang et al., 1993)where 1 gram of the ground frozen sample is added to 5 ml of extractionbuffer (2% hexadectltrimethylamminium bromide, 2% polyvilylpyrrolidone K30, 100 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2.0 M NaCl, 0.5 g/L spermidineand 2% beta-mercaptoethanol, previously warmed to 65° C.) and mixed byinversion and vortexing. The solution is extracted two times with anequal volume of chloroform:isoamyl alcohol and precipitated overnight at−20° C. after the addition of 0.25 volumes of 10M LiCl₂. Pellets arewashed with 70% EtOH, air dried and resuspended in RNase-free water.

EXAMPLE 2.2 Preparation of cDNA

Total RNA (5 μg) from each sample is reverse transcribed. First strandcDNA synthesis is accomplished at 42° C. for one hour using 5 μg oftotal RNA from Arabidopsis tissue, 100 pmol of an oligo dT(₂₄) primercontaining a 5′ T7 RNA polymerase promoter sequence[5′-GGCCAGTGAATTGTAATACGACTCACTATAGGGAGGCGG-(dT)₂₄-3′; SEQ ID NO:4710]synthesized by Genosys, and SUPERSCRIPT™ II reverse transcriptase (RT)(Gibco/BRL).

First strand cDNA synthesis reactions performed with SUPERSCRIPT™ II RTare carried out according to the manufacturer's recommendations using 50mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl₂, 10 mM dithiotreitol (DTT),0.5 mM dNTPs, and 200 units of RT enzyme.

The second cDNA strand is synthesized using 40 units of E. coli DNApolymerase I, 10 units of E. coli DNA ligase, and 2 units of RNase H ina reaction containing 25 mM Tris-HCl (pH 7.5), 100 mM KCl, 5 mM MgCl₂,10 mM (NH₄)SO₄, 0.15 mM β-NAD⁺, 1 mM dNTPs, and 1.2 mM DTT. The reactionproceeded at 16° C. for 2 hours and is terminated using EDTA.Double-stranded cDNA products are purified by phenol/chloroformextraction and ethanol precipitation.

EXAMPLE 2.3 Preparation of Biotinylated cRNA Probes

Synthesized cDNAs (approximately 0.1 μg) are used as templates toproduce biotinylated cRNA probes by in vitro transcription using T7 RNAPolymerase (ENZO BioArray High Yield RNA Transcript Labeling Kit).Labeled cRNAs are purified using affinity resin (Qiagen RNEASY® SpinColumns) and randomly fragmented to produce molecules of approximately35 to 200 bases. Fragmentation is achieved by incubation at 94° C. for35 minutes in a buffer containing 40 mM Tris-acetate, pH 8.1, 100 mMpotassium acetate, and 30 mM magnesium acetate.

EXAMPLE 2.4 Array Hybridization

The labeled samples are mixed with 0.1 mg/mL sonicated herring sperm DNAin a hybridization buffer containing 100 mM2-N-Morpholino-ethane-sulfonic acid (MES), 1 M NaCl, 20 mM EDTA, 0.01%Tween 20, denatured at 99° C. for 5 min, and equilibrated at 45° C. for5 min before hybridization. The hybridization mix is then transferred tothe Arabidopsis GeneChip genome array (Affymetrix) cartridge andhybridized at 45° C. for 16 h on a rotisserie at 60 rpm.

The hybridized arrays are then rinsed and stained in a fluidics station(Affymetrix). They are first rinsed with wash buffer A (6× SSPE (0.9 MNaCl, 0.06 M NaH₂PO₄, 0.006 M EDTA), 0.01% Tween 20, 0.005% Antifoam) at25° C. for 10 min and incubated with wash buffer B (100 mM MES, 0.1 MNaCl, 0.01% Tween 20) at 50° C. for 20 min, then stained withStreptavidin Phycoerythrin (SAPE) (100 mM MES, 1 M NaCl, 0.05% Tween 20,0.005% Antifoam, 10 mg/mL SAPE 2 mg/mL BSA) at 25° C. for 10 min, washedwith wash buffer A at 25° C. for 20 min and stained with biotinylatedanti-streptavidin antibody at 25° C. for 10 min. After staining, arraysare stained with SAPE at 25° C. for 10 min and washed with wash buffer Aat 30° C. for 30 min. The probe arrays are scanned twice and theintensities are averaged with a Hewlett-Packard GeneArray Scanner.

GeneSpring software was used to analyze relative expression levels andcompare tissue-specificity of gene expression.

EXAMPLE 2.5 Data Analysis

GENECHIP® Suite 3.2 (Affymetrix) is used for data normalization. Theoverall intensity of all probe sets of each array is scaled to 100 sohybridization intensity of all arrays is equivalent. False positives aredefined based on experiments in which samples are split, hybridized toGENECHIP® expression arrays and the results compared. A false positiveis indicated if a probe set is scored qualitatively as an “Increase” or“Decrease” and quantitatively as changing by at least two fold andaverage difference is greater than 25. A significant change is definedas 2-fold change or above with an expression baseline of 25, which isdetermined as the threshold level according to the scaling.

The expression data of selected genes are then normalized. Briefly, themedian of the expression level within each chip is calculated, and thedifference between the average difference and median average differenceis used as new value to measure the gene expression level. Theexpression data are also adjusted across different chip experimentsaccording to the calculated medium. Normalized data (genes and arrays)are analysed by the self organization map (SOM) method (Tamayo et al.,P.N.A.S., 96:2907 (1999), and then subject to heirachy cluster analysis(Eisen et al., P.N.A.S., 95:14863 (1998). By the cluster analysis, genesand chip experiments are clustered according to the expression levels.

2.5.1. Promoter Analysis

Generally, a database with rice contigs and Perl scripts were employedto determine which rice contig contained sequences from the identifiedgenes. Five gene prediction programs were analyzed on these contigs andthe rice sequence was blasted to these predictions. The prediction thatcontained the entire rice sequence within an exon was used to find thepromoter that was adjacent to the first exon.

For Oryza genes that were constitutively expressed, a cut off value of250 in all samples was used to screen for genes that were expressed inall tissues (range of 250-8638). The background level (gene notexpressed) was 50. This analysis resulted in the identification of 618genes that were constitutively expressed (Table 1A). The ORFs for 398 ofthose genes are listed in SEQ ID NOs:1-398 and the promoters for some ofthose genes in SEQ ID NOs:1598-1885 and 5960-5971, respectively. Basedon expression analysis, 150 genes were selected (Table 1B) and 120 genesof those considered for further analysis (Table 1C). Primers wereprepared to isolate 38 promoters from the 120 genes (Table 12).Preferred constitutively expressed genes include but are not limited tothose having SEQ ID NOs:7, 10, 12, 14, 22, 53, 54, 63, 84, 102, 103,123, 128, and 136, and orthologs thereof, e.g., promoters having SEQ IDNOs:1647, 1634, 1606, 1684, 1631, 1662, 1691, 1630, 1603, 1663, 1604, oran ortholog thereof. Further preferred constitutively expressed genesinclude but are not limited to those having SEQ ID NOs: 5928, 5929,5930, 5931, 5932, 5933, 5934, 5935, 5936, 5937, 5938, and 5939, andorthologs thereof, e.g., promoters having SEQ ID NOs: 5960, 5961, 5962,5963, 5964, 5965, 5966, 5967, 5968, 5969, 5970, and 5971, or an orthologthereof.

For Oryza genes expressed primarily in seed tissue, all genes that wereexpressed at 50 or above in at least one of the 29 tissues (about 13,450genes) were filtered to be expressed less than 50 in all non-seedrelated samples, not including aleurone, seed coat, embryo, endospermand seed milk, soft dough and hard dough. These analyses resulted in theidentification of 812 genes that were preferentially expressed in seedtissue (Table 2A). The ORFs for 578 of those genes are listed in SEQ IDNOs:1020-1567 and the promoters for some of those in SEQ IDNOs:2275-2672. Preferred seed-specific promoters are those from geneshaving SEQ ID NOs:1021-1023, 1028, 1044, 1033, 1068, 1403, 1081, 1048,1046, 1097, 1309, 1147, 1038, 1107, 1161, 1162, 1505, and 1026 and theorthologs thereof, e.g., promoters having SEQ ID NOs:2275-2277, 2279,2289, 2283, 2317, 2293, 2291, 2464, 2364, 2286, 2325, 2376, 2377, and2586, and an ortholog thereof. Further preferred are those from geneshaving SEQ ID NOs: 5927, 5940, 5941, and 5945-5958 and the orthologsthereof, e.g., promoters having SEQ ID NOs: , 5959, 5972, 5973,5977-5990 and 6001, and an ortholog thereof.

For seed-specific genes that were expressed only in a particular part ofa seed, e.g., embryo, endosperm, aleurone, genes that were expressed at50 or above in the particular sample but less than 50 in all othersamples absent that particular tissue sample were selected. Thus,embryo-specific, endosperm-specific and aleurone-specific genes wereidentified (Tables 3-5). Preferred aleurone-specific promoters are thosefrom genes having SEQ ID NOs:1045, 1165, 1324, 1150, 1547, 1373, and5927 and the orthologs thereof, e.g., promoters having SEQ ID NOs:2290,2380, 2366, 2627 and 5959, or an ortholog thereof. Preferredembryo-specific promoters are from genes having SEQ ID NOs:1294, 1346,1325, 1412, 1079 and the orthologs thereof, e.g., a promoter having SEQID NO:2315 or an ortholog thereof. Further preferred embryo-specificpromoters are from genes having SEQ ID NOs: 5940 and 5941 and theorthologs thereof, e.g., a promoter having SEQ ID NO: 5972 and 5973, oran ortholog thereof. Preferred endosperm-specific promoters are fromgenes having SEQ ID NOs:1043 and 1215 and the orthologs thereof, e.g., apromoter having SEQ ID NO:2411 or an ortholog thereof.

A cut off value of less than 50 in all non-root samples was used toscreen for Oryza genes that were expressed in a root-specific manner.The background level (gene not expressed) was 50. Genes that wereexpressed at greater than 50 in one or more of all root samples wereselected. This analysis resulted in the identification of 265 genes thatwere expressed primarily in root tissue (Table 8A). The ORFs for 219 ofthese genes is shown in SEQ ID NOs:801-1019 and some of the promoters inSEQ ID NOs:2144-2274.

For Oryza genes expressed primarily in Oryza panicle tissue (flower andpollen), all genes that were expressed at 50 or above on at least one ofthe rice panicle chips (about 10,597 genes) were filtered to beexpressed less than 50 in (i) leaf samples at germinating seed,tillering, mature and senescence stages; (ii) root samples atgerminating seed, tillering, booting, mature and panicle emergencestages; (iii) stem samples at panicle emergence and senescence stages;and (iv) seed hard dough and aleurone samples. These analyses resultedin the identification of 335 genes that were preferentially expressed inpanicle tissue (Table 7A). The ORFs for 256 of those genes is listed inSEQ ID NOs:465-720 and some of the promoters in SEQ ID NOs:1919-2085(for panicle). Preferred panicle-specific promoters are those from geneshaving SEQ ID NOs:689, 511, 482, 467 and 468, and the orthologs thereof,e.g., promoters having SEQ ID NOs:1920-1921, 2054, or an orthologthereof.

Eighty pollen-specific Oryza genes were identified (Table 9A and SEQ IDNOs:721-800) as well some pollen-specific promoters (SEQ IDNOs:2086-2143 Preferred pollen-specific promoter are those from geneshaving SEQ ID NOs:723-726 and 728 and the orthologs thereof, e.g.,promoters having SEQ ID NOs:2088-2090, or an ortholog thereof.

For Oryza genes expressed primarily in leaf and stem, i.e., greentissue, all genes that were expressed at 50 or above in at least threeof the tissues (about 12,563 genes) were filtered to be expressed morethan 50 in arial 3-4 leaf stage samples; less than 50 in all seedsamples (day 0-19); and less than 50 in aleurone, embryo, endosperm andpollen samples. Analysis revealed 90 genes expressed in arial tissue attillering stages that were expressed 2 fold greater than in root tissueat tillering stage (Table 6A). The ORFs for 66 of those genes are shownin SEQ ID NOs:399-464 and some of the promoters for those genes in SEQID NOs:1886-1918. Preferred green tissue-specific promoters are thosefrom genes having SEQ ID NOs:401, 405, 408, 410, 416, 417, 419, 433,438, 447 and 454 and orthologs thereof, e.g., promoters having SEQ IDNOs:1903, 1910, 1897, 1890, 1891, or an ortholog thereof. Leaf-specificbut not fruit-specific genes were determined by filtering the genes asfollows: relative expression of less than 50 in all of the seed samples,and greater than 50 in the leaf at tillering stage sample. This analysisresulted in the identification of five rice sequences: 5942/5991 (RF1;OS009452.1), OS012592.1, OS019946, OS001669.1, and OS002989.1. Thepromoter for one such gene is shown in SEQ ID NOs: 5974 and 5996,respectively.

EXAMPLE 3 Promoter Analysis

The gene chip experiment described above are designed to uncover genesthat are constitutively or tissue specifically (tissue-preferentially)expressed. Candidate promoters are identified based upon the expressionprofiles of the associated transcripts representatives of which areprovided in SEQ ID NOs: 1598-1885 and SEQ ID NOs: 1886-2672,respectively and further in SEQ ID Nos: 5960-5971, 5972-5990, and5996-6001.

Candidate promoters are obtained by PCR and fused to a GUS reporter genecontaining an intron. Both histochemical and fluormetric GUS assays arecarried out on stably transformed rice and maize plants and GUS activityis detected in the transformants.

Further, transient assays with the promoter: GUS constructs are carriedout in rice embryogenic callus and GUS activity is detected byhistochemical staining according the protocol described below (seeExample 12).

EXAMPLE 3.1 Construction of Binary Promoter: Reporter Plasmids

To construct a binary promoter: reporter plasmid for rice transformationa vector containing a candidate promoter of interest (i.e., the DNAsequence 5′ of the initiation codon for the gene of interest) is used,which results from recombination in a BP reaction between a PCR productusing the promoter of interest as a template and pDONR201™, producing anentry vector. The regulatory/promoter sequence is fused to the GUSreporter gene (Jefferson et al, 1987) by recombination using GATEWAY®Technology according to manufacturers protocol as described in theInstruction Manual (GATEWAY® Cloning Technology, GIBCO BRL, Rockville,Md., USA).

Briefly, the GATEWAY® Gus-intron-Gus (GIG)/NOS expression cassette isligated into pNOV2117 binary vector in 5′ to 3′ orientation. The 4.1 kBexpression cassette is ligated into the Kpn-I site of pNOV2117, thenclones are screened for orientation to obtain pNOV2346, a GATEWAY®adapted binary destination vector.

The promoter fragment in the entry vector is recombined via the LRreaction with the binary destination vector containing the GUS codingregion with an intron that has an attR site 5′ to the GUS reporter,producing a binary vector with a promoter fused to the GUS reporter(pNOVCANDProm). The orientation of the inserted fragment is maintainedby the att sequences and the final construct is verified by sequencing.The construct is then transformed into Agrobacterium tumefaciens strainsby electroporation as described herein below.

EXAMPLE 3.2 Transient Expression Analysis of Candidate Promoters in RiceEmbryogenic Callus 3.2.1 Materials:

-   -   Embryogenic rice callus (Kaybonett cultivar)    -   LBA 4404 Agrobacterium strains    -   KCMS liquid media for re-suspending bacterial pellet    -   200 mM stock (40 mg/ml) Acetosyringone    -   Sterile filter paper discs (8.5 mm in diameter)    -   LB spec liquid culture    -   MS-CIM media plates    -   MS-AS plates (co-cultivation plates)    -   MS-Tim plates (recovery plates)    -   Gus staining solution

3.2.2 Methods: 3.2.2.1 Induction of Embryogenic Callus:

-   -   1. Sterilize mature Kaybonett rice seeds in 40% ultra Clorox, 1        drop Tween 20, for 40 min.    -   2. Rinse with sterile water and plate on MS-CIM media (12        seeds/plate)    -   3. Grow in dark for four weeks.    -   4. Isolate embryogenic calli from scutellum to MS-CIM    -   5. Let grow in dark 8 days before use for transformation

3.2.2.2 Agrobacterium Preparation and Induction:

-   -   1. Start 6 mL shaking cultures of LBA4404 Agrobacterium strains        harboring rice promoter binary plasmids.    -   2. Grow the cultures at room temperature for 48 hrs in the        rotary shaker.    -   3. Spin down the cultures at 8,000 rpm at 4° C. and re-suspend        bacterial pellets in 10 ml of KCMS media supplemented with 100        μM Acetosyringone.    -   4. Place in the shaker at room temp for 1 hr for induction of        Agrobacterium virulence genes.    -   5. In a sterile hood dilute Agrobacterium cultures 1:3 in KSMS        media and transfer diluted cultures into deep petri dishes.

3.2.2.3 Inoculation of Plant Material and Staining:

-   -   6. In a sterile hood transfer embryogenic callus into diluted        Agrobacerium solution and incubate for 30 minutes.    -   7. In a sterile hood blot callus tissue on sterile filter paper        and transfer on MS-AS plates.    -   8. Co-culture plates in 22° C. growth chamber in the dark for        two days.    -   9. In a sterile hood transfer callus tissue to MS-Tim plates for        the tissue recovery (the presence of Timentin will prevent        Agrobacterium growth).    -   10. Incubate tissue on MS-Tim media for two days at 22° C. in        the dark.    -   11. Remove callus tissue from the plates and stain for 48 hrs.        in GUS staining solution.    -   12. De-stain tissue in 70% EtOH for 24 hours.

3.2.2.4 Recipies:

KCMS Media (Liquid), pH to 5.5

-   -   100 ml/l MS Major Salts, 10 ml/l MS Minor Salts, 5 ml/l MS iron        stock, 0.5M K₂HPO₄, 0.1 mg/ml Myo-Inositol, 1.3 μg/ml Thiamine,        0.2 g/ml 2,4-D (1 mg/ml), 0.1 g/ml Kinetin, 3% Sucrose, 100 μM        Acetosyringone

MS-CIM Media, pH 5.8

-   -   MS Basal salt (4.3 g/L), B5 Vitamins (200×) (5 m/L), 2% Sucrose        (20 g/L), Proline (500 mg/L), Glutamine (500 mg/L), Casein        Hydrolysate (300 mg/L), 2 μg/ml 2,4-D, Phytagel (3 g/L)

MS-As Medium, pH 5.8

-   -   MS Basal salt (4.3 g/L), B5 Vitamins (200×) (5 m/L), 2% Sucrose        (20 g/L), Proline (500 mg/L), Glutamine (500 mg/L), Casein        Hydrolysate (300 mg/L), 2 μg/ml 2,4-D, Phytagel (3 g/L), 200 μM        Acetosyringone

MS-Tim Media, pH 5.8

-   -   MS Basal salt (4.3 g/L), B5 Vitamins (200×) (5 m/L), 2% Sucrose        (20 g/L), Proline (500 mg/L), Glutamine (500 mg/L), Casein        Hydrolysate (300 mg/L), 2 μg/ml 2,4-D, Phytagel (3 g/L), 400        mg/l Timentin

Gus Staining Solution, pH 7

0.3M Mannitol; 0.02M EDTA, pH=7.0; 0.04 NaH₂PO₄; 1 mM x-gluc

The binary Promoter:Reporter Plasmids described in Example 3 above canalso be used for stable transformation of rice and maize plantsaccording to the protocols provided in Examples 10.1 and 10.2,respectively.

Pro- leaf moter SEQ Binary y leaf-2 root root-2 flower seed antherpollen Rice Name ID Vector leaf o leaf stem root husk kernel silk pollencomments Maize RC11 pNOV6043 − − + − − Rice RC11 pNOV6043 − + − + − −Maize baz, e, s RC2 5963 pNOV6030 −/+ + + + + + Rice RC2 5963 pNOV6030 −− −/+ − +baz − −!′ Maize RC26 5966 pNOV6046 − + − + − − Rice RC26 5966pNOV6046 − ++ − +baz − −! Maize RC33 5968 pNOV6044 − + −/+ + − − RiceRC33 5968 pNOV6044 − + − +baz, s, p − −! Maize RF1 5974 pNOV6045 − + − +− − Rice RF1 5974 pNOV6045 − + − +baz − − Maize RS10 5977 pNOV6034 − −/+−/+ − ++ Rice RS10 5977 pNOV6034 − + − ++e − −! Seed Maize specific,root back- ground RS18 6001 pNOV6035 −/+ −/+ −/+ −/+ −/+ +/− Rice RS186001 pNOV6035 − −/+ Maize RS3 2275 pNOV6031 − −/+ − + ++ ++ Rice RS32275 pNOV6031 − + − +baz − “−!” Seed Maize specific RS4 2276 pNOV6032 −−/+ −/+ − −/+ +++ Rice RS4 2276 pNOV6032 − − − +++ec − −! Seed Maizespecific RS8 2283 pNOV6033 +/− −/+ − −/+ −/+ +++ ++ − Rice RS8 2283pNOV6033 − −/+ − ++e, p − − Seed Maize specific ZmU pNOV6048 ++ +++ ++++ Positive Rice BIintron Control ZmU pNOV6048 ++ +++ +++ +++ ++ +++Positive Maize BIintron Control GUS staining scores: + = stainingobserved ++ = strong staining +++ = very strong staining − = no staining“−!” = some pollen grains stained, probably contamination +/− = somelines showed staining, but not all −/+ = staining faint or seen in only1 or 2 lines blank = not analyzed yet Note: many RS seem to stain inkernel and root Key: baz-black abscission zone s-scutellum p-pedicele-endosperm or embryo sc-seed coat

EXAMPLE 4 Rice Orthologs of Arabidopsis Tissue-Specifically ExpressedGenes Identified by Reverse Genetics

Understanding the function of every gene is the major challenge in theage of completely sequenced eukaryotic genomes. Sequence homology can behelpful in identifying possible functions of many genes. However,reverse genetics, the process of identifying the function of a gene byobtaining and studying the phenotype of an individual containing amutation in that gene, is another approach to identify the function of agene.

Reverse genetics in Arabidopsis has been aided by the establishment oflarge publicly available collections of insertion mutants (Krysan etal., (1999) Plant Cell 11, 2283-2290; Tisser et al., (1999) Plant Cell11, 1841-1852; Speulman et al., (1999) . Plant Cell 11, 1853-1866;Parinov et al., (1999). Plant Cell 11, 2263-2270; Parinov andSundaresan, 2000; Biotechnology 11, 157-161). Mutations in genes ofinterest are identified by screening the population by PCR amplificationusing primers derived from sequences near the insert border and the geneof interest to screen through large pools of individuals. Poolsproducing PCR products are confirmed by Southern hybridization andfurther deconvoluted into subpools until the individual is identified(Sussman et al., (2000) Plant Physiology 124, 1465-1467).

Recently, some groups have begun the process of sequencing insertionsite flanking regions from individual plants in large insertion mutantpopulations, in effect prescreening a subset of lines for genomicinsertion sites (Parinov et al., (1999) . Plant Cell 11, 2263-2270;Tisser et al., (1999) . Plant Cell 11, 1841-1852). The advantage to thisapproach is that the laborious and time-consuming process of PCR-basedscreening and deconvolution of pools is avoided.

A large database of insertion site flanking sequences from approximately100,000 T-DNA mutagenized Arabidopsis plants of the Columbia ecotype(GARLIC lines) is prepared. T-DNA left border sequences from individualplants are amplified using a modified thermal asymmetricinterlaced-polymerase chain reaction (TAIL-PCR) protocol (Liu et al.,(1995). Plant J. 8, 457-463). Left border TAIL-PCR products aresequenced and assembled into a database that associates sequence tagswith each of the approximately 100,000 plants in the mutant collection.Screening the collection for insertions in genes of interest involves asimple gene name or sequence BLAST query of the insertion site flankingsequence database, and search results point to individual lines.Insertions are confirmed using PCR.

Analysis of the GARLIC insert lines suggests that there are 76,856insertions that localize to a subset of the genome representing codingregions and promoters of 22,880 genes. Of these, 49,231 insertions liein the promoters of over 18,572 genes, and an additional 27,625insertions are located within the coding regions of 13,612 genes.Approximately 25,000 T-DNA left border mTAIL-PCR products (25% of thetotal 102,765) do not have significant matches to the subset of thegenome representing promoters and coding regions, and are thereforepresumed to lie in noncoding and/or repetitive regions of the genome.The Arabidopsis T-DNA GARLIC insertion collection is used to investigatethe roles of certain genes, which are expressed in specific planttissues. Target genes are chosen using a variety of criteria, includingpublic reports of mutant phenotypes, RNA profiling experiments, andsequence similarity to tissue-specific genes. Plant lines withinsertions in genes of interest are then identified. Each T-DNAinsertion line is represented by a seed lot collected from a plant thatis hemizygous for a particular T-DNA insertion. Plants homozygous forinsertions of interest are identified using a PCR assay. The seedproduced by these plants is homozygous for the T-DNA insertion mutationof interest.

Homozygous mutant plants are tested for altered grain composition. Thegenes interrupted in these mutants contribute to the observed phenotype.

Rice orthologs of the Arabidopsis genes are identified by similaritysearching of a rice database using the Double-Affine Smith-Watermanalgorithm (BLASP with e values better than⁻¹⁰).

EXAMPLE 5 Cloning and Sequencing of Nucleic Acid Molecules from Rice

5.1 Genomic DNA: Plant genomic DNA samples are isolated from acollection of tissues which are listed in Table 1. Individual tissuesare collected from a minimum of five plants and pooled. DNA can beisolated according to one of the three procedures, e.g., standardprocedures described by Ausubel et al. (1995), a quick leaf prepdescribed by Klimyuk et al. (1993), or using FTA paper (LifeTechnologies).

For the latter procedure, a piece of plant tissue such as, for example,leaf tissue is excised from the plant, placed on top of the FTA paperand covered with a small piece of parafilm that serves as a barriermaterial to prevent contamination of the crushing device. In order todrive the sap and cells from the plant tissue into the FTA paper matrixfor effective cell lysis and nucleic acid entrapment, a crushing deviceis used to mash the tissue into the FTA paper. The FTA paper is airdried for an hour. For analysis of DNA, the samples can be archived onthe paper until analysis. Two mm punches are removed from the specimenarea on the FTA paper using a 2 mm Harris MICRO-PUNCH™ and placed intoPCR tubes. Two hundred (200) microliters of FTA purification reagent isadded to the tube containing the punch and vortexed at low speed for 2seconds. The tube is then incubated at room temperature for 5 minutes.The solution is removed with a pipette so as to repeat the wash one moretime. Two hundred (200) microliters of TE (10 mM Tris, 0.1 mM EDTA, pH8.0) is added and the wash is repeated two more times. The PCR mix isadded directly to the punch for subsequent PCR reactions.

5.2 Cloning of Candidate cDNA: A candidate cDNA is amplified from totalRNA isolated from rice tissue after reverse transcription using primersdesigned against the computationally predicted cDNA. Primers designedbased on the genomic sequence can be used to PCR amplify the full-lengthcDNA (start to stop codon) from first strand cDNA prepared from ricecultivar Nipponbare tissue.

The Qiagen RNEASY® kit (Qiagen, Hilden, Germany) is used for extractionof total RNA. The SUPERSCRIPT™ II kit (Invitrogen, Carlsbad, Calif.,USA) is used for the reverse transcription reaction. PCR amplificationof the candidate cDNA is carried out using the reverse primer sequencelocated at the translation start of the candidate gene in 5′-3′direction. This is performed with high-fidelity Taq polymerase(Invitrogen, Carlsbad, Calif., USA).

The PCR fragment is then cloned into PCR® 2.1-TOP® (Invitrogen) or thePGEM®-T easy vector (Promega Corporation, Madison, Wis., USA) per themanufacturer's instructions, and several individual clones are subjectedto sequencing analysis.

5.3 DNA sequencing: DNA preps for 2-4 independent clones are minipreppedfollowing the manufacturer's instructions (Qiagen). DNA is subjected tosequencing analysis using the BIGDYE™ Terminator Kit according tomanufacturer's instructions (ABI). Sequencing makes use of primersdesigned to both strands of the predicted gene of interest. DNAsequencing is performed using standard dye-terminator sequencingprocedures and automated sequencers (models 373 and 377; AppliedBiosystems, Foster City, Calif.). All sequencing data are analyzed andassembled using the Phred/Phrap/Consed software package (University ofWashington) to an error ratio equal to or less than 10⁻⁴ at theconsensus sequence level.

The consensus sequence from the sequencing analysis is then to bevalidated as being intact and the correct gene in several ways. Thecoding region is checked for being full length (predicted start and stopcodons present) and uninterrupted (no internal stop codons). Alignmentwith the gene prediction and BLAST analysis is used to ascertain thatthis is in fact the right gene.

The clones are sequenced to verify their correct amplification.

EXAMPLE 6 Functional Analysis in Plants

A plant complementation assay can be used for the functionalcharacterization of the tissue-specifically expressed genes according tothe invention.

Rice and Arabidopsis putative orthologue pairs are identified usingBLAST comparisons, TFASTXY comparisons, and Double-Affine Smith-Watermansimilarity searches. Constructs containing a rice cDNA or genomic cloneinserted between the promoter and terminator of the Arabidopsisorthologue are generated using overlap PCR (Gene 77, 61-68 (1989)) andGATEWAY® cloning (Life Technologies Invitrogen). For ease of cloning,rice cDNA clones are preferred to rice genomic clones. A three stage PCRstrategy is used to make these constructs.

(1) In the first stage, primers are used to PCR amplify: (i) 2Kbupstream of the translation start site of the Arabidopsis orthologue,(ii) the coding region or cDNA of the rice orthologue, and (iii) the 500bp immediately downstream of the Arabidopsis orthogue's translation stopsite. Primers are designed to incorporate onto their 5′ ends at least 16bases of the 3′ end of the adjacent fragment, except in the case of themost distal primers which flank the gene construct (the forward primerof the promoter and the reverse primer of the terminator). The forwardprimer of the promoters contains on their 5′ ends partial AttB1 sites,and the reverse primer of the terminators contains on their 5′ endspartial AttB2 sites, for GATEWAY® cloning.

(2) In the second stage, overlap PCR is used to join either the promoterand the coding region, or the coding region and the terminator.

(3) In the third stage either the promoter-coding region product can bejoined to the terminator or the coding region-terminator product can bejoined to the promoter, using overlap PCR and amplification with fulllAtt site-containing primers, to link all three fragments, and put fullAtt sites at the construct termini.

The fused three-fragment piece flanked by GATEWAY® cloning sites areintroduced into the LTI donor vector pDONR201 using the BP clonasereaction, for confirmation by sequencing. Confirmed sequenced constructsare introduced into a binary vector containing GATEWAY® cloning sites,using the LR clonase reaction such as, for example, pAS200.

The pAS200 vector was created by inserting the GATEWAY® cloning cassetteRfA into the Acc65I site of pNOV3510.

pNOV3510 was created by ligation of inverted pNOV2114 VSI binary intopNOV3507, a vector containing a PTX5′ Arab Protox promoter driving thePPO gene with the Nos terminator.

pNOV2114 was created by insertion of virGN54D (Pazour et al. 1992, J .Bacteriol. 174:4169-4174) from pAD1289 (Hansen et al. 1994, PNAS91:7603-7607) into pHiNK085.

pHiNK085 was created by deleting the 35S:PMI cassette and M13 ori inpVictorHiNK.

pPVictorHiNK was created by modifying the T-DNA of pVictor (described inWO 97/04112) to delete M13 derived sequences and to improve its cloningversatility by introducing the BIGLINK polylinker.

The sequence of the pVictor HiNK vector is disclosed in SEQ ID NO: 5 inWO 00/6837, which is incorporated herein by reference. The pVictorHiNKvector contains the following constituents that are of functionalimportance:

-   -   The origin of replication (ORI) functional in Agrobacterium is        derived from the Pseudomonas aeruginosa plasmid pVS1 (Itoh et        al. 1984. Plasmid 11: 206-220; Itoh and Haas, 1985. Gene 36:        27-36). The pVS1 ORI is only functional in Agrobacterium and can        be mobilised by the helper plasmid pRK2013 from E. coli into A.        tumefaciens by means of a triparental mating procedure (Ditta et        al., 1980. Proc. Natl. Acad. Sci USA 77: 7347-7351).    -   The ColE1 origin of replication functional in E. coli is derived        from pUC19 (Yannisch-Perron et al., 1985. Gene 33: 103-119).    -   The bacterial resistance to spectinomycin and streptomycin        encoded by a 0.93 kb fragment from transposon Tn7 (Fling et        al., 1985. Nucl. Acids Res. 13: 7095) functions as selectable        marker for maintenance of the vector in E. coli and        Agrobacterium. The gene is fused to the tac promoter for        efficient bacterial expression (Amman et al., 1983. Gene 25:        167-178).    -   The right and left T-DNA border fragments of 1.9 kb and 0.9 kb        that comprise the 24 by border repeats, have been derived from        the Ti-plasmid of the nopaline type Agrobacterium tumefaciens        strains pTiT37 (Yadav et al., 1982. Proc. Natl. Acad. Sci. USA.        79: 6322-6326).

The plasmid is introduced into Agrobacterium tumefaciens GV3101pMP90 byelectroporation. The positive bacterial transformants are selected on LBmedium containing 50 μg/μl kanamycin and 25 μg/μl gentamycin. Plants aretransformed by standard methodology (e.g., by dipping flowers into asolution containing the Agrobacterium) except that 0.02% Silwet-77(Lehle Seeds, Round Rock, Tex., USA) is added to the bacterialsuspension and the vacuum step omitted. Five hundred (500) mg of seedsare planted per 2 ft² flat of soil and and progeny seeds are selectedfor transformants using PPO selection.

Primary transformants are analyzed for complementation. Primarytransformants are genotyped for the Arabidopsis mutation and presence ofthe transgene. When possible, >50 mutants harboring the transgene shouldbe phenotyped to observe variation due to transgene copy number andexpression.

EXAMPLE 7 Vector Construction for Overexpression and Gene “Knockout”Experiments

7.1 Overexpression

Vectors used for expression of full-length “candidate genes” of interestin plants (overexpression) are designed to overexpress the protein ofinterest and are of two general types, biolistic and binary, dependingon the plant transformation method to be used.

For biolistic transformation (biolistic vectors), the requirements areas follows:

-   -   1. a backbone with a bacterial selectable marker (typically, an        antibiotic resistance gene) and origin of replication functional        in Escherichia coli (E. coil; eg. ColE1), and    -   2. a plant-specific portion consisting of:        -   a. a gene expression cassette consisting of a promoter (eg.            ZmUBIint MOD), the gene of interest (typically, a            full-length cDNA) and a transcriptional terminator (eg.            Agrobacterium tumefaciens nos terminator);        -   b. a plant selectable marker cassette, consisting of a            promoter (eg. rice Act1D-BV MOD), selectable marker gene            (eg. phosphomannose isomerase, PMI) and transcriptional            terminator (eg. CaMV terminator).            Vectors designed for transformation by Agrobacterium            tumefaciens (A. tumefaciens; binary vectors) consist of:    -   1. a backbone with a bacterial selectable marker functional in        both E. coli and A. tumefaciens (eg. spectinomycin resistance        mediated by the aadA gene) and two origins of replication,        functional in each of aforementioned bacterial hosts, plus        the A. tumefaciens virG gene;    -   2. a plant-specific portion as described for biolistic vectors        above, except in this instance this portion is flanked by A.        tumefaciens right and left border sequences which mediate        transfer of the DNA flanked by these two sequences to the plant.

7.2 Knock Out Vectors

Vectors designed for reducing or abolishing expression of a single geneor of a family or related genes (knockout vectors) are also of twogeneral types corresponding to the methodology used to downregulate geneexpression: antisense or double-stranded RNA interference (dsRNAi).

(a) Anti-Sense

For antisense vectors, a full-length or partial gene fragment(typically, a portion of the cDNA) can be used in the same vectorsdescribed for full-length expression, as part of the gene expressioncassette. For antisense-mediated down-regulation of gene expression, thecoding region of the gene or gene fragment will be in the oppositeorientation relative to the promoter; thus, mRNA will be made from thenon-coding (antisense) strand in planta.

(b) dsRNAi

For dsRNAi vectors, a partial gene fragment (typically, 300 to 500basepairs long) is used in the gene expression cassette, and isexpressed in both the sense and antisense orientations, separated by aspacer region (typically, a plant intron, eg. the OsSH1 intron 1, or aselectable marker, eg. conferring kanamycin resistance). Vectors of thistype are designed to form a double-stranded mRNA stem, resulting fromthe basepairing of the two complementary gene fragments in planta.

Biolistic or binary vectors designed for overexpression or knockout canvary in a number of different ways, including eg. the selectable markersused in plant and bacteria, the transcriptional terminators used in thegene expression and plant selectable marker cassettes, and themethodologies used for cloning in gene or gene fragments of interest(typically, conventional restriction enzyme-mediated or GATEWAY®recombinase-based cloning). An important variant is the nature of thegene expression cassette promoter driving expression of the gene or genefragment of interest in most tissues of the plants (constitutive, eg.ZmUBIint MOD), in specific plant tissues (eg. maize ADP-gpp forendosperm-specific expression), or in an inducible fashion (eg.GAL4bsBz1 for estradiol-inducible expression in lines constitutivelyexpressing the cognate transcriptional activator for this promoter).

EXAMPLE 8 Insertion of a “Candidate Gene” into Expression Vector

A validated rice cDNA clone in PCR® 2.1-TOPO® or the PGEM®-T easy vectoris subcloned using conventional restriction enzyme-based cloning into avector, downstream of the maize ubiquitin promoter and intron, andupstream of the Agrobacterium tumefaciens nos 3′ end transcriptionalterminator. The resultant gene expression cassette (promoter, “candidategene” and terminator) is further subcloned, using conventionalrestriction enzyme-based cloning, into the pNOV2117 binary vector(Negrotto et al (2000) Plant Cell Reports 19, 798-803; plasmid pNOV117disclosed in this article corresponds to pNOV2117 described herein),generating pNOVCAND.

The pNOVCAND binary vector is designed for transformation andover-expression of the “candidate gene” in monocots. It consists of abinary backbone containing the sequences necessary for selection andgrowth in Escherichia coli DH-5α (Invitrogen) and Agrobacteriumtumefaciens LBA4404 (pAL4404; pSB1), including the bacterialspectinomycin antibiotic resistance aadA gene from E. coli transposonTn7, origins of replication for E. coli (ColE1) and A. tumefaciens(VS1), and the A. tumefaciens virG gene. In addition to the binarybackbone, which is identical to that of pNOV2114 described hereinpreviously (see Example 5 above), pNOV2117 contains the T-DNA portionflanked by the right and left border sequences, and including thePOSITECH™ (Syngenta) plant selectable marker (WO 94/20627) and the“candidate gene” gene expression cassette. The POSITECH™ plantselectable marker confers resistance to mannose and in this instanceconsists of the maize ubiquitin promoter driving expression of the PMI(phosphomannose isomerase) gene, followed by the cauliflower mosaicvirus transcriptional terminator.

Plasmid pNOV2117 is introduced into Agrobacterium tumefaciens LBA4404(pAL4404; pSB1) by electroporation. Plasmid pAL4404 is a disarmed helperplasmid (Ooms et al (1982) Plasmid 7, 15-29). Plasmid pSB1 is a plasmidwith a wide host range that contains a region of homology to pNOV2117and a 15.2 kb KpnI fragment from the virulence region of pTiBo542(Ishida et al (1996) Nat Biotechnol 14, 745-750). Introduction ofplasmid pNOV2117 into Agrobacterium strain LBA4404 results in aco-integration of pNOV2117 and pSB1.

Alternatively, plasmid pCIB7613, which contains the hygromycinphosphotransferase (hpt) gene (Gritz and Davies, Gene 25, 179-188, 1983)as a selectable marker, may be employed for transformation.

Plasmid pCIB7613 (see WO 98/06860, incorporated herein by reference inits entirety) is selected for rice transformation. In pCIB7613, thetranscription of the nucleic acid sequence codinghygromycin-phosphotransferase (HYG gene) is driven by the corn ubiquitinpromoter (ZmUbi) and enhanced by corn ubiquitin intron 1. The3′polyadenylation signal is provided by NOS 3′ nontranslated region.

Other useful plasmids include pNADII002 (GAL4-ER-VP16) which containsthe yeast GAL4 DNA Binding domain (Keegan et al., Science, 231:699(1986)), the mammalian estrogen receptor ligand binding domain (Greeneet al., Science, 231 :1150 (1986)) and the transcriptional activationdomain of the HSV VP16 protein (Triezenberg et al., 1988). Both hpt andGAL4-ER-VP16 are constitutively expressed using the maize Ubiquitinpromoter, and pSGCDL1 (GAL4BS Bz1 Luciferase), which carries the fireflyluciferase reporter gene under control of a minimal maize Bronze1 (Bz1)promoter with 10 upstream synthetic GAL4 binding sites. All constructsuse termination signals from the nopaline synthase gene.

EXAMPLE 9 Plant Transformation EXAMPLE 9.1 Rice Transformation

pNOVCAND is transformed into a rice cultivar (Kaybonnet) usingAgrobacterium-mediated transformation, and mannose-resistant calli areselected and regenerated.

Agrobacterium is grown on YPC solid plates for 2-3 days prior toexperiment initiation. Agrobacterial colonies are suspended in liquid MSmedia to an OD of 0.2 at λ600 nm. Acetosyringone is added to theagrobacterial suspension to a concentration of 200 μM and agro isinduced for 30 min.

Three-week-old calli which are induced from the scutellum of matureseeds in the N6 medium (Chu, C. C. et al., Sci, Sin., 18, 659-668(1975))are incubated in the agrobacterium solution in a 100×25 petri plate for30 minutes with occasional shaking. The solution is then removed with apipet and the callus transfered to a MSAs medium which is overlayed withsterile filter paper.

Co-Cultivation is continued for 2 days in the dark at 22° C.

Calli are then placed on MS-Timetin plates for 1 week. After that theyare tranfered to PAA+mannose selection media for 3 weeks.

Growing calli (putative events) are picked and transfered to PAA+mannosemedia and cultivated for 2 weeks in light.

Colonies are tranfered to MS20SorbKinTim regeneration media in platesfor 2 weeks in light. Small plantlets are transferred to MS20SorbKinTimregeneration media in GA7 containers. When they reach the lid, they aretransfered to soil in the greenhouse.

Expression of the “candidate gene” in transgenic T_(o) plants isanalyzed. Additional rice cultivars, such as but not limited to,Nipponbare, Taipei 309 and Fuzisaka 2 are also transformed and assayedfor expression of the “candidate gene” product and enhanced proteinexpression.

EXAMPLE 9.2 Maize Transformation

Transformation of immature maize embryos is performed essentially asdescribed in Negrotto et al., (2000) Plant Cell Reports 19: 798-803. Forthis example, all media constituents are as described in Negrotto etal., supra. However, various media constituents described in theliterature may be substituted.

9.2.1. Transformation Plasmids and Selectable Marker

The genes used for transformation are cloned into a vector suitable formaize transformation as described in Example 17. Vectors used containthe phosphomannose isomerase (PMI) gene (Negrotto et al. (2000) PlantCell Reports 19: 798-803).

9.2.2. Preparation of Agrobacterium Tumefaciens

Agrobacterium strain LBA4404 (pSB1) containing the plant transformationplasmid is grown on YEP (yeast extract (5 g/L), peptone (10 g/L), NaCl(5 g/L), 15 g/l agar, pH 6.8) solid medium for 2 to 4 days at 28° C.Approximately 0.8×10⁹ Agrobacteria are suspended in LS-inf mediasupplemented with 100 μM acetosyringone (As) (Negrotto et al.,(2000)Plant Cell Rep 19: 798-803). Bacteria are pre-induced in this medium for30-60 minutes.

9.2.3. Inoculation

Immature embryos from A188 or other suitable maize genotypes are excisedfrom 8-12 day old ears into liquid LS-inf+100 μM As. Embryos are rinsedonce with fresh infection medium. Agrobacterium solution is then addedand embryos are vortexed for 30 seconds and allowed to settle with thebacteria for 5 minutes. The embryos are then transferred scutellum sideup to LSAs medium and cultured in the dark for two to three days.Subsequently, between 20 and 25 embryos per petri plate are transferredto LSDc medium supplemented with cefotaxime (250 mg/l) and silvernitrate (1.6 mg/l) and cultured in the dark for 28° C. for 10 days.

9.2.4. Selection of Transformed Cells and Regeneration of TransformedPlants

Immature embryos producing embryogenic callus are transferred toLSD1M0.5S medium. The cultures are selected on this medium for 6 weekswith a subculture step at 3 weeks. Surviving calli are transferredeither to LSD1M0.5S medium to be bulked-up or to Reg1 medium. Followingculturing in the light (16 hour light/8 hour dark regiment), greentissues are then transferred to Reg2 medium without growth regulatorsand incubated for 1-2 weeks. Plantlets are transferred to Magenta GA-7boxes (Magenta Corp, Chicago, Ill., USA) containing Reg3 medium andgrown in the light. Plants that are PCR positive for thepromoter-reporter cassette are transferred to soil and grown in thegreenhouse.

EXAMPLE 10 Chromosomal Markers to Identify the Location of a NucleicAcid Sequence

The sequences of the present invention can also be used for SSR mapping.SSR mapping in rice has been described by Miyao et al. (DNA Res 3:233(1996)) and Yang et al. (Mol Gen Genet 245:187 (1994)), and in maize byAhn et al. (Mol Gen Genet 241:483 (1993)). SSR mapping can be achievedusing various methods. In one instance, polymorphisms are identifiedwhen sequence specific probes flanking an SSR contained within asequence are made and used in polymerase chain reaction (PCR) assayswith template DNA from two or more individuals or, in plants, nearisogenic lines. A change in the number of tandem repeats between theSSR-flanking sequence produces differently sized fragments (U.S. Pat.No. 5,766,847). Alternatively, polymorphisms can be identified by usingthe PCR fragment produced from the SSR-flanking sequence specific primerreaction as a probe against Southern blots representing differentindividuals (Refseth et al., Electrophoresis 18:1519 (1997)). Rice SSRscan be used to map a molecular marker closely linked to functional gene,as described by Akagi et al. (Genome 39:205 (1996)).

The sequences of the present invention can be used to identify anddevelop a variety of microsatellite markers, including the SSRsdescribed above, as genetic markers for comparative analysis and mappingof genomes.

Many of the polynucleotides listed in Tables 2 to 11 contain at least 3consecutive di-, tri- or tetranucleotide repeat units in their codingregion that can potentially be developed into SSR markers. Trinucleotidemotifs that can be commonly found in the coding regions of saidpolynucleotides and easily identified by screening the polynucleotidessequences for said motifs are, for example: CGG; GCC, CGC, GGC, etc.Once such a repeat unit has been found, primers can be designed whichare complementary to the region flanking the repeat unit and used in anyof the methods described below.

Sequences of the present invention can also be used in a variation ofthe SSR technique known as inter-SSR (ISSR), which uses microsatelliteoligonucleotides as primers to amplify genomic segments different fromthe repeat region itself (Zietkiewicz et al., Genomics 20:176 (1994)).ISSR employs oligonucleotides based on a simple sequence repeat anchoredor not at their 5′- or 3′-end by two to four arbitrarily chosennucleotides, which triggers site-specific annealing and initiates PCRamplification of genomic segments which are flanked by inverselyorientated and closely spaced repeat sequences. In one embodiment of thepresent invention, microsatellite markers as disclosed herein, orsubstantially similar sequences or allelic variants thereof, may be usedto detect the appearance or disappearance of markers indicating genomicinstability as described by Leroy et al. (Electron. J Biotechnol, 3(2),(2000), available at the website of the Electronic Journal ofBiotechnology), where alteration of a fingerprinting pattern indicatedloss of a marker corresponding to a part of a gene involved in theregulation of cell proliferation. Microsatellite markers are useful fordetecting genomic alterations such as the change observed by Leroy etal. (Electron. J Biotechnol, 3(2), supra (2000)) which appeared to bethe consequence of microsatellite instability at the primer binding siteor modification of the region between the microsatellites, andillustrated somaclonal variation leading to genomic instability.Consequently, sequences of the present invention are useful fordetecting genomic alterations involved in somaclonal variation, which isan important source of new phenotypes.

In addition, because the genomes of closely related species are largelysyntenic (that is, they display the same ordering of genes within thegenome), these maps can be used to isolate novel alleles from wildrelatives of crop species by positional cloning strategies. This sharedsynteny is very powerful for using genetic maps from one species to mapgenes in another. For example, a gene mapped in rice providesinformation for the gene location in maize and wheat.

EXAMPLE 11 Quantitative Trait Linked Breeding

Various types of maps can be used with the sequences of the invention toidentify Quantitative Trait Loci (QTLs) for a variety of uses, includingmarker-assisted breeding. Many important crop traits are quantitativetraits and result from the combined interactions of several genes. Thesegenes reside at different loci in the genome, often on differentchromosomes, and generally exhibit multiple alleles at each locus.Developing markers, tools, and methods to identify and isolate the QTLsinvolved in a trait, enables marker-assisted breeding to enhancedesirable traits or suppress undesirable traits. The sequences disclosedherein can be used as markers for QTLs to assist marker-assistedbreeding. The sequences of the invention can be used to identify QTLsand isolate alleles as described by Li et al. in a study of QTLsinvolved in resistance to a pathogen of rice. (Li et al., Mol Gen Genet261:58 (1999)). In addition to isolating QTL alleles in rice, othercereals, and other monocot and dicot crop species, the sequences of theinvention can also be used to isolate alleles from the correspondingQTL(s) of wild relatives. Transgenic plants having various combinationsof QTL alleles can then be created and the effects of the combinationsmeasured. Once an ideal allele combination has been identified, cropimprovement can be accomplished either through biotechnological means orby directed conventional breeding programs. (Flowers et al., J Exp Bot51:99 (2000); Tanksley and McCouch, Science 277:1063 (1997)).

EXAMPLE 12 Marker-Assisted Breeding

Markers or genes associated with specific desirable or undesirabletraits are known and used in marker assisted breeding programs. It isparticularly beneficial to be able to screen large numbers of markersand large numbers of candidate parental plants or progeny plants. Themethods of the invention allow high volume, multiplex screening fornumerous markers from numerous individuals simultaneously.

Markers or genes associated with specific desirable or undesirabletraits are known and used in marker assisted breeding programs. It isparticularly beneficial to be able to screen large numbers of markersand large numbers of candidate parental plants or progeny plants. Themethods of the invention allow high volume, multiplex screening fornumerous markers from numerous individuals simultaneously.

A multiplex assay is designed providing SSRs specific to each of themarkers of interest. The SSRs are linked to different classes of beads.All of the relevant markers may be expressed genes, so RNA or cDNAtechniques are appropriate. RNA is extracted from root tissue of 1000different individual plants and hybridized in parallel reactions withthe different classes of beads. Each class of beads is analyzed for eachsample using a microfluidics analyzer. For the classes of beadscorresponding to qualitative traits, qualitative measures of presence orabsence of the target gene are recorded. For the classes of beadscorresponding to quantitative traits, quantitative measures of geneactivity are recorded. Individuals showing activity of all of thequalitative genes and highest expression levels of the quantitativetraits are selected for further breeding steps. In procedures wherein noindividuals have desirable results for all the measured genes,individuals having the most desirable, and fewest undesirable, resultsare selected for further breeding steps. In either case, progeny arescreened to further select for homozygotes with high quantitative levelsof expression of the quantitative traits.

EXAMPLE 13 Method of Modifying the Gene Frequency

The invention further provides a method of modifying the frequency of agene in a plant population, including the steps of: identifying an SSRwithin a coding region of a gene; screening a plurality of plants usingthe SSR as a marker to determine the presence or absence of the gene inan individual plant; selecting at least one individual plant forbreeding based on the presence or absence of the gene; and breeding atleast one plant thus selected to produce a population of plants having amodified frequency of the gene. The identification of the SSR within thecoding region of a gene can be accomplished based on sequence similaritybetween the nucleic acid molecules of the invention and the regionwithin the gene of interest flanking the SSR.

EXAMPLE 14 Testing of Rice Promoters In Plants

The promoters of Table 13 were cloned from an entry vector using theGATEWAY® system into the pNOV2346 vector pNOV2346 had the promoter ofinterest controlling the GUS gene and ZmUBI controlling the PMI genewith a NOS terminator. All constructs were submitted to CC and thentransformed into rice or maize. Most were only transformed in maize. 10to 15 single copy events were chosen for T0 analysis. All tissue typeswere sampled for two assays. For rice; leaf, root, flower and seed. Formaize; leaf, root, husk, silk, tassel, stem, pollen and kernel. Thefirst assay was GUS histochemical staining. The tissues were put into asolution containing ImM X-Gluc overnight at 37° C. The tissues werescored based on staining results. Most often a GUS fluorometric assaywas done on the tissues to quantify the GUS expression. After the assayswere done, pictures were taken of all the tissues. The pictures and theGUS fluorometric assay results were uploaded to the promoters database.

In addition, T1 analysis was done on some constructs in maize. 10-15homozygous plants were chosen and the same assays were performedaccording to the following protocols.

GUS Staining Protocol

-   -   1. Sample tissue into well of tissue culture plate.    -   2. Add 50 μL of Silwett L-77 to 500 mL of GUS stain (pH 7.5 for        Arab, pH 8.0 for rice and maize).    -   3. Add GUS stain to the wells until the tissue is covered.    -   4. Incubate at 37 degrees Celsius for 48 hours, 1.5 hr for rice        and maize seed.    -   5. Destain in 70% EtOH, change everyday if needed.

GUS Fluorometric Assay Protocol Solution Preparations:

-   -   1. Prepare Extraction Buffer—EB (Recipe 1) in advance in large        volumes and store at +4° C.    -   2. Prepare Assay Buffer (Recipe 2) in EB in large volumes and        store at −80° C. up to 6 months in the dark (wrap tubes in foil        or tape).        -   Important! Do not thaw and refreeze repeatedly as this will            lead to loss of activity.    -   3. Prepare Stop (2% Na₂CO₃) Solution (Recipe 3) in large volume        and store at room temperature    -   4. Prepare 1 mM 4-MU Storage Stock and 10 μM 4-MU Working Stock        (Recipe 4)    -   5. Prepare 24× standards of BSA in Extraction buffer (Recipe 6)        and keep at +4° C.

Tissue Harvest:

Organ and tissue harvest may vary with each organ, tissue type.

-   -   1. Harvest appropriate amount of tissue into 96 well block with        metal beads    -   2. Following harvest tissue may be processed immediately or deep        freezed on dry ice or in liquid N₂ and stored at −80° C. until        processing.

Extraction:

-   -   1. Grind tissue in bead beater, Kleco or SawsAll for 2 min    -   2. Freeze block on dry ice or liquid nitrogen o/n    -   3. That block and repeat grinding as in step 1    -   4. Add 250 μl of chilled GUS EB        -   You will need a minimum of 200 μl for each sample, but will            usually use 0.3-1.0 ml/sample. Adjust volume according to            amount of tissue being ground. Try to use the least amount            of buffer that is possible to prevent diluting activity            below limits of detection.    -   5. Following tissue grinding, clear extracts by centrifuging        blocks for 10 min at +4° C. at 4000 RPM (EPPENDORF® 5810 R).    -   6. Remove supernatant into a fresh 96 well block, keep        supernatant (plant extract) on ice at all times    -   7. Following extraction samples can be either:        -   for long term storage: frozen in liquid nitrogen and stored            at −80° C. up to 6 months        -   or for a short term storage: can be stored at +4° C. up to 1            week prior to carrying out assays        -   Warning! Do no keep your extracts frozen at −20° C. as this            will kill enzymatic activity in your extracts

MUG Assay

-   1. Thaw appropriate number of tubes with Assay buffer (see Recipe    2). One tube is usually enough for one 96 well plate.-   2. Prepare assay plates by adding 250 μl of Assay Buffer in the same    # of wells as samples and pre-warm the assay plates at +37° C. for    at least 30 min-   3. Prepare Stop 96 well black, clear flat bottom Plates (greiner    bio-one stock 655096) by adding 270 μl Stop Solution (2% Na₂CO₃)    leaving the last 12th column empty for the 4-MU standard (added    later)    -   Important! It is recommended that separate Stop Plates should be        used for each individual time point (required for easier        transfer of data from the plate reader to the MUG and BCA Macro)-   4. Add 50 μl of extracts to pre-warmed assay plates (this is the    incubation mix)

For High Expressing Promoters:

-   5. Immediately after adding extracts to the Assay block remove 30 μl    of the Incubation mix and add to the Stop plate (this is your 0 min    time point)-   6. Begin timing incubation as soon as extract is added to the assay    buffer and remove 30 μl of samples to stop plates at 15 min, 30 min    and 60 min time points

For Low and Medium Expressing Promoters:

-   7. As soon as extracts were added to Assay Blocks start    pre-Incubation period for 30 min at +37° C.-   8. Following 30 min pre-incubation time remove 30 μl of incubation    mix to stop buffer at 0 min, 30 min and 60 min time points

Measure Fluorescence on Plate Reader

-   9. Make 4-MU standards 0, 50, 100, 250, 500, 1000, 1500, 2000 pmol    MU diluted in 2% Na₂CO₃ (Recipe 5) and place standards in the last    12th column in each assay plate-   10. Measure activity on Tecan Spectrafluor Plus with 360 nm    excitation and 465 nm emission (readings done with 3 flashes and    gain optimized to give measurable readings for all samples—typically    40-60)    -   Activity can be read immediately (preferred) or hold samples        overnight at room temperature and measure activity the following        day.-   11. Transfer readings into MUGletc sheets of MUG and BCA Macro for    Gus Activity calculations

BCA Assays:

-   12. Prepare 1 to 2 dilution plates for roots:    -   Alternatively, 25 μl of undiluted extract can be used fro the        BCA    -   Add 50 μl of extract in new 96 well plate and mix with 50 μl of        water    -   Add 50 μl of 2× BSA Standards in tne last 12th column of the        same plate and add 50 μl of water-   13. Prepare 1 to 12 dilution plates for all other tissues:    -   Remove 40 μl of 1:2 dilutions of extracts and BSA Standards and        add 200 μl of water-   14. Prepare BCA—Assay plates;    -   Add 25 μl of appropriate dilutions into 96 well clear flat        bottom plates (greiner bio-one stock 655101), last column is        filled with appropriate dilutions of BSA standards with the        final concentrations of: 0, 25, 50. 75, 100, 150, 200, 250 μg/ml    -   Add 100 μl of BCA reagent (Recipe 7)-   15. Incubate BCA-Assay plates at +37° C. for 30 min-   16. Cool down BCA-Assay plates at room temperature for 10 min-   17. Read samples immediately on Tecan Spectrafluor Plus with 560 nm    Absorbance filter-   18. Transfer readings into BCA1 sheet of MUG and BCA Macro for Gus    Activity calculations

Recipe 1: GUS Extraction Buffer-EB

50 mM NaPO4 pH 7.0; 1 mMDDT; 10 mM EDTA; 0.1% Sarcosyl; 0.1% Triton

Stock: For 100 ml For 500 ml For 1000 ml 1M NaPO4 pH 7.0 5.0 ml 25.0 ml50 ml 1M DTT in H2O 0.1 ml 0.5 ml 1 ml 0.5M Na2ESTA 2 ml 10 ml 20 ml 20%Sarcosyl 0.5 ml 2.5 ml 5 ml 10% Triton 1.0 ml 5.0 ml 10 ml Dd H2O 91.4ml 457 ml 914 ml

Recipe 2: Assay Buffer

Prepare Assay buffer by dissolving appropriate amount of4-Methylumbelliferyl β-D-Glucuronide (Sigma M-9130) in appropriateamount of EB. Generally, make large volume of assay buffer. Aliquot 25ml, 15 ml or smaller volume in new 15, 30 or 50 ml or any other tube,wrap them in foil or tape and freeze immediately. If kept frozen at −80°C. and not thawed and re-freezed the substrate is stable for severalmonths (up to 6 months). Typically the left over unused assay bufferfrom one tube is discarded.

Compound For 50 ml EB For 100 ml EB For 200 ml EB For 300 ml EB For 500ml EB 4-Methylumbelliferyl 17.62 mg 35.23 mg 70.46 mg 105.69 mg 176.15mg β-D-Glucuronide

Assay Buffer Number of Aliquot samples 1.5 ml  6 5 ml 20 10 ml 40 15 ml60 25 ml  84 (1 block) 50 ml 168 (2 blocks)

Recipe 3: Stop Solution-2% Na₂CO₃

Dissolve appropriate amount of Na₂CO₃ in water

Compound For 100 ml For 1 L For 2 L For 5 L Na₂CO₃ 2 g 20 g 40 g 100 g

Recipe 4: Storage 1 mM 4-MU Stock and Working 10 μM 4-MU Stock

Dissolve appropriate amount of 4-Methylumbelliferone ( ) (Fluka 69580)in water. This Storage Stock solution can be stored at +4° C. in thedark (wrap container in foil) up to 3 months

Compound For 10 ml For 50 ml For 100 ml 4-Methylumbelliferone 1.94 mg9.7 mg 19.4 mg ( )

To make a Working 10 μM 4-MU Stock (10000 nmol/ml or 1000 pmoles/100 μl)dilute the appropriate amount of 1 mM 4-MU stock in 2% Na₂CO₃. Thissolution can be stored at +4° C. in the dark (wrap container in foil) nolonger than 1 month.

Solution For 10 ml For 15 ml For 30 ml 4-Methylumbellifeone 100 μl 150μl 300 μl ( )

Recipe 5: 4-MU Standard Curve Preparation

-   1. Make samples for standard curve by mixing appropriate amount of    10 μM 4-MU Working Stock with the appropriate amount of Stop buffer.    Total volume for each dilution should be 300 μl

Volume of 10 μM Volume of 2% pmol 4-MU 4-MU working stock Na₂CO₃ 0  0 μl300 μl 50  5 μl 295 μl 100  10 μl 290 μl 250  25 μl 275 μl 500  50 μl250 μl 1000 100 μl 200 μl 1500 150 μl 150 μl 2000 200 μl 100 μl

Recipe 6: 24× BSA Solutions in Extraction Buffer

Dissolve an appropriate amount of BSA (Pierce or Sigma) in appropriatevolume of extraction buffer. The stocks can be stored at +4° C.

24× Stock Concentration Amount of Amount of Amount of μg/ml BSA for 10ml BSA for 15 ml BSA for 30 ml 0  0 mg  0 mg  0 mg 600  6 mg  9 mg  18mg 1200 12 mg 18 mg  36 mg 1800 18 mg 27 mg  54 mg 2400 24 mg 36 mg  72mg 3600 36 mg 54 mg 108 mg 4800 48 mg 72 mg 144 mg 6000 60 mg 90 mg 180mg

Recipe 7: BOA Reagent

Add 1 ml of BCA Protein Assay Reagent B (Pierce #23224) into 50 ml ofBCA Protein Assay reagent A (Pierce #23221), mix well

Results showed that RAI was a low-expressing promoter, as was RC17. RC22was pollen and kernel preferred with low expression in roots. RC9 was alow constitutive expresser. REM13-1573 showed embryo-preferredexpression, as did REM7. RENS showed endosperm-specific expression, andRGT1 showed green tissue-preferred expression. RR1 proved to be aroot-preferred expresser, as did RR2. RS13 showed seed-preferredexpression, as did RS15. RS25, RS26, and RS3 were very low expressingpromoters. RS4 was found to be a strong endosperm-preferred promoterwith some residual activity in root stem and husk. RS5 wasseed-preferred, and RS6 showed strong expression in roots. RS8 showedendosperm-preferred expression, with some residual activity in root andtassels/flowers. RT16 and RT27 are transcription factors.

Supporting TABLES

Lengthy table referenced here US20110225678A1-20110915-T00001 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110225678A1-20110915-T00002 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110225678A1-20110915-T00003 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110225678A1-20110915-T00004 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110225678A1-20110915-T00005 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110225678A1-20110915-T00006 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110225678A1-20110915-T00007 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110225678A1-20110915-T00008 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110225678A1-20110915-T00009 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110225678A1-20110915-T00010 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110225678A1-20110915-T00011 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110225678A1-20110915-T00012 Pleaserefer to the end of the specification for access instructions.

Lengthy table referenced here US20110225678A1-20110915-T00013 Pleaserefer to the end of the specification for access instructions.

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

LENGTHY TABLES The patent application contains a lengthy table section.A copy of the table is available in electronic form from the USPTO website(http://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20110225678A1).An electronic copy of the table will also be available from the USPTOupon request and payment of the fee set forth in 37 CFR 1.19(b)(3).

1. An isolated promoter comprising the polynucleotide sequence of SEQ IDNO: 6086, wherein the polynucleotide sequence is operatively linked to aheterologous nucleic acid.
 2. An expression vector comprising a promotercomprising the nucleic acid sequence of SEQ ID NO: 6086 operably linkedto a heterologous nucleic acid, wherein the promoter directs expressionof the heterologous nucleic acid in a plant cell.
 3. A plant celltransformed with the expression vector of claim
 2. 4. A transgenic plantcomprising the plant cell of claim
 3. 5. A polynucleotide comprising aplant nucleotide sequences that directs root specific expression of anoperatively linked nucleic acid segment in a plant cell, wherein saidpolynucleotide sequence is at least 90% identical to promoter RR1 (SEQID NO: 6086).